WO2022161371A1 - 体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法 - Google Patents

体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法 Download PDF

Info

Publication number
WO2022161371A1
WO2022161371A1 PCT/CN2022/073892 CN2022073892W WO2022161371A1 WO 2022161371 A1 WO2022161371 A1 WO 2022161371A1 CN 2022073892 W CN2022073892 W CN 2022073892W WO 2022161371 A1 WO2022161371 A1 WO 2022161371A1
Authority
WO
WIPO (PCT)
Prior art keywords
flow channel
circulating tumor
tumor cells
sorting
channel
Prior art date
Application number
PCT/CN2022/073892
Other languages
English (en)
French (fr)
Inventor
蒙玄
杨家敏
Original Assignee
广州万孚生物技术股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州万孚生物技术股份有限公司 filed Critical 广州万孚生物技术股份有限公司
Priority to US18/263,397 priority Critical patent/US20240084233A1/en
Priority to EP22745245.5A priority patent/EP4286506A1/en
Publication of WO2022161371A1 publication Critical patent/WO2022161371A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • the invention relates to the technical field of cell sorting, in particular to an in vitro analysis and diagnosis instrument, a microfluidic chip and a method for sorting and enriching circulating tumor cells.
  • Cancer is the second most common cause of death globally, accounting for 1 in 6 deaths.
  • Tumor metastasis is the cause of 90% of cancer deaths.
  • the process of tumor metastasis is the shedding of tumor cells from the primary tumor or metastases, circulating in the lymphatic system or peripheral blood, invading distant tissues, and forming new tumor foci, eventually leading to the death of the patient. These shed tumor cells are called circulating tumor cells (CTC). Therefore, by examining the number and types of circulating tumor cells in the blood, the dynamic changes of tumor lesions can be monitored and the treatment effect can be evaluated.
  • the isolated circulating tumor cells can be analyzed by immunophenotyping and genome sequencing to find drug targets, thereby realizing personalized and precise treatment. Circulating tumor cells are also extremely important for early screening of tumors.
  • tumors are 1-2 mm in size, imaging and other methods are difficult to detect. But in the early stages of many cancers, the blood contains a certain number of circulating tumor cells. For some high-risk patients, regular circulating tumor cell detection is helpful for early detection of cancer, early treatment, and prevention of disease progression.
  • sorting and enriching circulating tumor cells from blood is particularly important, but the content of circulating tumor cells is extremely low, usually 1-10 circulating tumor cells per milliliter of blood, and hundreds of white blood cells per milliliter. There are billions of red blood cells, and it is difficult to sort and enrich circulating tumor cells like finding a needle in a haystack.
  • the techniques for sorting and enriching circulating tumor cells are roughly divided into: immunomagnetic beads method, density gradient centrifugation method, membrane filtration method, and microfluidic chip technology. Among them, circulating tumor cells and leukocytes in the flow channel of the traditional microfluidic chip easily overlap, which is not conducive to the recovery of circulating tumor cells.
  • a microfluidic chip for sorting and enriching circulating tumor cells comprising a functional board, the first side of the functional board is provided with:
  • a primary selection flow channel communicated with the sample inlet, and used to initially aggregate circulating tumor cells and leukocytes in the sample
  • the fine screening flow channel is communicated with the primary selection flow channel, and the side of the fine screening flow channel away from the accumulation of circulating tumor cells is dug with a deepened flow channel, and the deepened flow channel is arranged along the extension direction of the fine screening flow channel And the depth of the deepening flow channel is greater than the depth of the fine screening flow channel.
  • the above-mentioned microfluidic chip for sorting and enriching circulating tumor cells dilutes the blood sample and passes it through the sample inlet into the primary selection flow channel. Due to the influence of inertial lift, Dean drag force, etc., the diameter of red blood cells is small, and the diameter of red blood cells is small.
  • the chaotic flow in the primary selection channel, and the large diameter of leukocytes and circulating tumor cells will initially aggregate into bands under the balance of forces in the primary selection channel, and then flow into the fine screening channel, and the circulating tumor cells will just aggregate.
  • the body is a thin band and is close to the bottom of the inner wall of the fine sieve flow channel, and the leukocytes have not yet accumulated at the bottom of the inner wall of the tube, but the bands of leukocytes and circulating tumor cells are close to each other.
  • the liquid flow state changes the inertial lift force and Dean drag force, destroying the original balance, so that the leukocytes can produce a disordered movement state, which in turn makes the leukocytes distribute more uniformly in the fine sieving flow channel, and avoids leukocyte aggregates.
  • Circulating tumor cells overlap, and red blood cells are distributed more evenly, which not only ensures that the aggregates of circulating tumor cells do not interfere, but also prevents leukocytes from accumulating at the bottom of the inner wall of the fine screening flow channel, which facilitates the subsequent separation of circulating tumor cells and leukocytes. Facilitates the recovery of circulating tumor cells.
  • the depth of the deepened flow channel is 50 ⁇ m-200 ⁇ m greater than the depth of the fine screening flow channel; or the depth of the deepened flow channel is greater than the depth of the fine screening flow channel by 70 ⁇ m-120 ⁇ m.
  • the fine screening flow channel is designed so that the ratio of the size of the circulating tumor cells to the hydraulic diameter is less than or equal to 0.5; or the fine screening flow channel is designed so that the ratio of the size of the circulating tumor cells to the hydraulic diameter is less than or equal to 0.07; or the fine screening flow channel is designed so that the ratio of the size of the circulating tumor cells to the hydraulic diameter is 0.045-0.065; or the fine screening flow channel is designed so that the ratio of the size of the circulating tumor cells to the hydraulic diameter is 0.05 to 0.06.
  • the first side surface of the functional plate is further provided with a first turning flow channel, the curvature radius of the first turning flow channel is larger than the curvature radius of the fine screen flow channel, and the first turning flow channel is The curved flow channel communicates with the fine screening flow channel, and the deepened flow channel correspondingly extends into the first curved flow channel and is located on the side away from the accumulation of circulating tumor cells.
  • a removal flow channel is further provided on the first side of the functional plate, the removal flow channel is communicated with an end of the first turning flow channel away from the fine screen flow channel, and the deepened flow channel Correspondingly extending into the removal flow channel and located on the side away from the accumulation of circulating tumor cells, the removal flow channel is provided with a shunt hole penetrating the wall surface of the removal flow channel.
  • the first side surface of the functional board is further provided with a second turning flow channel, the curvature radius of the second turning flow channel is larger than the curvature radius of the removal flow channel, and the second turning flow channel is The flow channel communicates with one end of the removal flow channel away from the first curved flow channel, and the deepened flow channel correspondingly extends into the second curved flow channel and is located on a side away from the accumulation of circulating tumor cells.
  • an end of the second turning flow channel away from the removal flow channel is provided with a recovery flow channel and a waste liquid flow channel that are independent of each other, and the recovery flow channel is close to the second turning flow channel.
  • the side where the circulating tumor cells gather is communicated, and the waste fluid flow channel is communicated with the side of the second curved flow channel that is close to the deepened flow channel.
  • the ratio of the liquid flowing out of the waste liquid flow channel and the recovery flow channel is 45%-65%: 3%-20%; or the waste liquid flow channel and the recovery flow
  • the proportion of the fluid flowing out of the canal is 50%-60%: 5%-10%.
  • the waste liquid flow channel and the recovery flow channel are in a reciprocating and folded structure.
  • the second side surface of the functional board is provided with a buffer flow channel in a reciprocating and folded structure, and the buffer flow channel communicates with the distribution hole.
  • the proportion of the liquid flowing out of the buffer flow channel is 30%-70%; or the proportion of the liquid flowing out of the buffer flow channel is 45%-60%.
  • a blocking member is provided in the removal flow channel corresponding to the inlet of the distribution hole, and the blocking member is located on the side of the distribution hole away from the deepened flow channel, and the blocking member is located along the The width in the extension direction of the removal flow channel is larger than the diameter of the distribution hole.
  • a plurality of shunt holes are arranged in sequence along the extending direction of the removal flow channel, and a plurality of buffer flow channels in a reciprocating and folded structure are arranged on the second side of the functional plate.
  • the channels are in one-to-one correspondence with the shunt holes; a blocking member is provided in the removal channel corresponding to the inlet of the shunt hole, and the blocking member is located on the side of the shunt hole away from the deepened flow channel, and the The width of the blocking member along the extending direction of the removal channel is larger than the diameter of the distribution hole.
  • the distances between the plurality of the distribution holes and the corresponding deepened flow channels gradually increase.
  • the length of the buffer flow channel corresponding to the diverter hole close to the first turning flow channel is greater than the length of other buffer flow channels.
  • the primary selection flow channel includes an introduction section, a connection section and a sorting section that are communicated in sequence, an end of the introduction section away from the connection section is communicated with the sample inlet, and the introduction section is reciprocating Foldback structure.
  • the connecting section includes a first straight pipe section, a first arc section, a second straight pipe section, a second arc section and a third straight pipe section that are communicated in sequence, and the first straight pipe section is connected to the lead-in section. One end of the section away from the sample inlet is connected, and the third straight pipe section is connected with the sorting section.
  • the end of the introduction section is arranged in a clockwise direction and is connected with the first straight pipe section.
  • the sorting section includes a connecting section and a main pipe section that are connected in sequence, the connecting section is connected to the third straight pipe section, the main section is connected to the fine screen flow channel, and the connecting section is connected to the fine screen flow channel.
  • the width of the segment perpendicular to its extension direction is a
  • the width of the main pipe segment perpendicular to its extension direction is b
  • a ⁇ b the sorting segment is an asymmetric wave channel in the width direction.
  • the fine-screening flow channel and the removal flow channel are sinusoidal arc-shaped flow channels.
  • the microfluidic chip for sorting and enriching circulating tumor cells further includes an upper cover plate and a lower cover plate, the upper cover plate is provided with a sample inlet, and the upper cover plate and The first side surface of the function board is stacked and connected, the lower cover plate is provided with a recovery hole, a waste liquid hole and a discharge hole, and the lower cover board is stacked and connected to the second side surface of the function board, so The recovery flow channel is communicated with the recovery hole, the waste liquid flow channel is communicated with the waste liquid hole, and the distribution hole is communicated with the discharge hole.
  • a method for sorting and enriching circulating tumor cells comprising the following steps:
  • the diluted blood sample is introduced through the sample inlet of the microfluidic chip.
  • the circulating tumor cells and leukocytes in the blood sample initially aggregate, and the circulating tumor cells aggregate into The thin band is close to the bottom of the inner wall of the primary selection flow channel, and the white blood cells have not yet gathered at the bottom of the inner wall of the primary selection flow channel, and the red blood cells are scattered in the primary selection flow channel;
  • the blood sample in the primary selection flow channel is introduced into the fine screening flow channel, the side of the fine screening flow channel away from the accumulation of circulating tumor cells is dug with a deepened flow channel, and the circulating tumor cells in the blood sample are aggregated into thin strips. It is close to the bottom of the inner wall of the fine screening flow channel, while the leukocytes are in a disordered movement state and are far away from the bottom of the inner wall of the fine screening flow channel.
  • the blood sample is diluted and passed through the sample inlet into the primary selection flow channel. Due to the influence of inertial lift, Dean drag force, etc., the diameter of red blood cells is small, and the diameter of red blood cells is small in the primary selection flow.
  • the chaotic flow in the channel, and the large diameter of leukocytes and circulating tumor cells will initially aggregate into bands under the balance of the forces in the primary channel, and then flow into the refining channel, and the circulating tumor cells will rigidly aggregate into fine particles.
  • the band is close to the bottom of the inner wall of the fine sieving channel, and the leukocytes have not yet accumulated at the bottom of the inner wall of the channel, but the bands where the leukocytes and circulating tumor cells gather are very close, by digging the side of the fine sieving channel away from the accumulation of circulating tumor cells.
  • a deepening flow channel is provided, and the deepening flow channel is arranged along the extension direction of the fine screening flow channel and the depth of the deepening flow channel is greater than that of the fine screening flow channel, which disrupts the liquid flow state near the outer wall of the fine screening flow channel , which changes the inertial lift force and Dean drag force and destroys the original balance, so that the leukocytes can produce a disordered movement state, which in turn makes the distribution of leukocytes in the fine sieve flow channel more uniform, avoiding leukocyte aggregates and circulating tumor cells.
  • the red blood cells are also distributed more evenly, which not only ensures that the aggregation of circulating tumor cells does not interfere, but also prevents leukocytes from accumulating at the bottom of the inner wall of the fine screening flow channel, which is convenient for the subsequent separation of circulating tumor cells and leukocytes, which is beneficial to circulating tumors. Recovery of cells.
  • the method for sorting and enriching circulating tumor cells further comprises the following steps:
  • the blood sample in the fine screening flow channel is introduced into the first turning flow channel with a radius of curvature greater than that of the fine screening flow channel, and the circulating tumor cells in the blood sample are aggregated into thin bands and further approach the bottom of the inner wall of the first turning flow channel ;
  • the blood sample in the first turning flow channel is introduced into a removal channel with a shunt hole, and the red blood cells and white blood cells in the blood sample flow out of the removal channel through the shunt hole, while the circulating tumor cells in the blood sample aggregate into Thin strip and close to the bottom of the inner wall of the removal channel;
  • the remaining blood sample after the removal of the flow channel is introduced into a second curved flow channel with a radius of curvature greater than that of the removed flow channel, and the circulating tumor cells in the blood sample are aggregated into thin strips and further approach the bottom of the inner wall of the second curved flow channel ;
  • One side of the second turning flow channel close to the accumulation of circulating tumor cells is connected with a recovery flow channel for recovering the blood sample on this side, and the other side is connected with a waste fluid flow channel and collects the blood sample on this side.
  • the method for sorting and enriching circulating tumor cells further comprises the following steps:
  • the blood sample recovered from the recovery channel is passed into the sample inlet of the microfluidic chip again, and the foregoing steps are repeated.
  • the method for sorting and enriching circulating tumor cells wherein a blocking member is provided in the removal channel corresponding to the inlet of the shunt hole, and the blocking member is located away from the shunt hole.
  • the width of the blocking member along the extension direction of the removal flow channel is greater than the diameter of the shunt hole, and the proportion of blood samples flowing out through the shunt hole is 30%-70%; The proportion of blood samples flowing out of the waste liquid flow channel is 45%-65%.
  • An in vitro analysis and diagnosis instrument comprising a main body and the microfluidic chip for sorting and enriching circulating tumor cells described in any one of the above, wherein the microfluidic chip for sorting and enriching circulating tumor cells can be matched with the main body use.
  • the main body is provided with a chip mounting position for installing the microfluidic chip for sorting and enriching circulating tumor cells, and a mixing chamber for mixing the sample, diluent and lysate evenly and a recovery cavity for recycling circulating tumor cells, the mixing cavity can be communicated with the sample inlet of the microfluidic chip for sorting and enriching circulating tumor cells, and the recycling cavity can be sorting and enriching with the circulating tumor cells.
  • the microfluidic chip is connected to the recovery pores flowing out of circulating tumor cells.
  • the in-vitro analysis and diagnosis instrument further includes a power system and a control system, the control system is used to control the power system to inject the sample, the diluent and the lysate into the mixing chamber in a specific proportion, and to inject the mixing chamber into the mixing chamber.
  • the mixed liquid of the body is passed into the microfluidic chip for sorting and enriching the circulating tumor cells at a certain flow rate.
  • the in-vitro analysis and diagnosis instrument further includes a first control valve
  • the main body is further provided with a cleaning fluid chamber, a sample chamber, a diluting fluid chamber and a lysing fluid chamber.
  • the liquid cavity, the sample cavity, the diluent cavity and the lysate cavity are respectively connected with the power system, and one end of the first control valve is connected to the cleaning liquid cavity, the sample cavity, the diluent cavity and the The outlet of the lysate chamber is connected, and the other end is connected to the mixing chamber.
  • the in vitro analysis and diagnosis instrument further includes a second control valve, one end of the second control valve is connected to the mixing chamber, and the other end is connected to the recovery chamber.
  • the cavity is also connected to the power system.
  • FIG. 1 is a schematic diagram of an upper cover plate of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment
  • FIG. 2 is a schematic diagram of a first side of a functional plate of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment
  • FIG. 3 is a schematic diagram of a second side of a functional plate of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment
  • FIG. 4 is a schematic diagram of a lower cover plate of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment
  • Figure 5 is a schematic cross-sectional view along section line A-A in Figure 2;
  • Fig. 6 is a schematic diagram of the motion state of circulating tumor cells, white blood cells and red blood cells in the blood sample in the introduction section and the connecting section of the primary selection flow channel;
  • FIG. 7 is a schematic diagram of the motion state of circulating tumor cells, white blood cells, and red blood cells in the blood sample in the sorting section of the primary selection flow channel;
  • FIG. 8 is a schematic diagram of the motion state of circulating tumor cells, white blood cells, and red blood cells in the blood sample in the first turning channel;
  • FIG. 9 is a schematic diagram of the movement state of circulating tumor cells, white blood cells and red blood cells in the blood sample in the removal channel;
  • Fig. 10 is a schematic diagram of the movement state of circulating tumor cells, white blood cells and red blood cells in the blood sample in the second turning channel;
  • FIG. 11 is a schematic diagram of an in vitro analysis and diagnostic apparatus in an embodiment.
  • an embodiment provides a microfluidic chip for sorting and enriching circulating tumor cells, including a functional board 1 . Further, the microfluidic chip for sorting and enriching circulating tumor cells further includes an upper cover plate 2 and a lower cover plate 3 .
  • the upper cover 2 is stacked and connected to the first side of the functional board 1
  • the lower cover 3 is stacked and connected to the second side of the functional board 1
  • the upper cover 2 is provided with a sample Entrance 21.
  • the first side surface of the functional board 1 is provided with a primary selection flow channel 10 and a fine screening flow channel 20 .
  • the primary selection flow channel 10 communicates with the sample inlet 21 , and is used to preliminarily aggregate the circulating tumor cells 03 and leukocytes 02 in the sample.
  • the fine screening flow channel 20 is communicated with the primary selection flow channel 10.
  • the side of the fine screening flow channel 20 away from the accumulation of circulating tumor cells 03 is dug with a deepened flow channel 30, and the deepened flow channel 30 runs along the fine screen.
  • the screen flow channel 20 is arranged in the extending direction, and the depth of the deepened flow channel 30 is greater than the depth of the fine screen flow channel 20 .
  • the sample injection hole 11 penetrates through the first side surface and the second side surface of the function board 1 , and is arranged corresponding to the sample inlet 21 . Due to the influence of inertial lift force, Dean drag force, etc., referring to FIG. 6 , the diameter of the red blood cells 01 in the sample is small, and they flow chaotically in the primary flow channel 10 , while the leukocytes 02 and circulating tumor cells 03 have a small diameter.
  • the diameter is larger, and under the balance of the force in the primary selection flow channel 10, it will initially gather into a band and then flow into the fine screening flow channel 20.
  • the circulating tumor cells 03 are rigidly aggregated into thin bands, and close to The bottom of the inner wall of the fine screening flow channel 20, and the leukocytes 02 have not yet accumulated at the bottom of the inner wall of the flow channel, but the bands where the leukocytes 02 and the circulating tumor cells 03 are gathered are very close to each other.
  • the deepening flow channel 30 is arranged along the extension direction of the fine screening flow channel 20, and the depth of the deepening flow channel 30 is greater than the depth of the fine screening flow channel 20, which disrupts the fine screen flow channel 20.
  • the liquid flow state near the outer wall of the sieve flow channel 20 changes the inertial lift and Dean drag force, destroying the original balance, so that the leukocytes O2 can produce a disordered movement state, and then make the leukocytes O2 in the fine sieve flow channel 20
  • the distribution is more uniform, avoiding the overlap of the leukocyte 02-mer with the circulating tumor cell 03, and the red blood cell 01 is also distributed more evenly, which not only ensures that the aggregate of circulating tumor cells does not interfere, but also prevents the leukocyte 02 from accumulating in the fine screen.
  • the bottom of the inner wall of the flow channel 20 is convenient for the subsequent separation of the circulating tumor cells 03 from the white blood cells 02 , which is conducive to the recovery of the circulating tumor cells 03 .
  • the fine screening flow channel is an asymmetrical wave-shaped channel with alternating large turns and small turns. In other embodiments, the fine screening flow channel can also be set as a straight channel.
  • the fine-screening flow channel is an asymmetrical wavy channel with a rectangular cross-section whose aspect ratio varies over the length of the channel, resulting in the formation of a single stream of aggregated particles.
  • the aspect ratio of the rectangular section varies from 8 to 30. In another embodiment, the aspect ratio of the rectangular section varies between 11-23.
  • the fine screening flow channel is designed such that the ratio of the size of the circulating tumor cells to the hydraulic diameter is less than or equal to 0.5.
  • the fine screening flow channel is designed so that the ratio of the size of the circulating tumor cells to the hydraulic diameter is less than or equal to 0.07.
  • the fine screening flow channel is designed so that the ratio of the size of the circulating tumor cells to the hydraulic diameter is 0.045-0.065.
  • the fine screening flow channel is designed so that the ratio of the size of the circulating tumor cells to the hydraulic diameter is 0.05-0.06.
  • the hydraulic diameter Dh is defined as 2wh/(w+h), referring to Figures 5 and 7; w and h are the width and height of the flow channel.
  • the first side surface of the functional board 1 is further provided with a first turning flow channel 40 .
  • the radius of curvature of the first turning flow channel 40 is greater than the radius of curvature of the fine screening flow channel 20, and the first turning flow channel 40 communicates with the fine screening flow channel 20.
  • the deepening flow channel 30 correspondingly extends into the first turning channel 40 and is located on the side away from the accumulation of circulating tumor cells 03 . Under the action of deepening the flow channel 30 , the distribution of the white blood cells 02 in the first curved flow channel 40 is more uniform, avoiding overlapping with the circulating tumor cells 03 .
  • a first turning flow channel 40 is connected to the fine screening flow channel 20 , and due to the large radius of the first turning flow channel 40 , the relative advantage of inertial lift is even greater.
  • the fluid in the middle of the channel is subjected to the largest centrifugal force, and thus flows to the outer edge of the channel.
  • the fluid velocity near the channel wall is the smallest, and the centrifugal force is also the smallest, so it is squeezed by the intermediate fluid. Therefore, under the action of the first curved flow channel 40, the circulating tumor cells 03 can get close to the bottom of the inner wall of the channel to the greatest extent, so as to prepare for the subsequent sorting of the circulating tumor cells 03 and the discharge of the leukocytes 02.
  • the depth of the deepening flow channel 30 is greater than the depth of the fine screening flow channel 20 by 50 ⁇ m-200 ⁇ m.
  • the depth of the fine screening flow channel is 100 ⁇ m-200 ⁇ m.
  • the depth of the deepening flow channel 30 is greater than the depth of the fine screening flow channel 20 by 70 ⁇ m-120 ⁇ m.
  • the depth of the fine screening flow channel is 110 ⁇ m-150 ⁇ m.
  • FIG. 5 is a schematic cross-sectional view of the deepening flow channel 30 in the first turning flow channel 40, wherein the fine screening flow channel 20 extends to connect with the first turning flow channel 40, and the first turning flow channel 40 also has a corresponding deepening flow channel. 30.
  • the depth difference H between the deepening flow channel 30 and the first turning flow channel 40 is 50 ⁇ m-200 ⁇ m, preferably 70 ⁇ m-120 ⁇ m. It can not only ensure that the circulating tumor cells O3 can flow as close to the bottom of the inner wall of the flow channel as possible in the fine screening flow channel 20 or the first turning flow channel 40, and at the same time disturb the liquid flow state on the side of the deepening flow channel 30, so as to avoid leukocyte O2 aggregation.
  • the primary selection flow channel 10 includes an introduction section 110 , a connection section 120 and a sorting section 130 that are communicated in sequence, and an end of the introduction section 110 away from the connection section 120 is connected to
  • the sample inlet 21 is in communication, and the introduction section 110 is in a reciprocating and folded structure.
  • red blood cells 01 , white blood cells 02 and circulating tumor cells 03 are evenly distributed in the flow channel.
  • the diameter of erythrocytes 01 is about 6-8 ⁇ m
  • the diameter of white blood cells 02 is about 8-12 ⁇ m
  • the diameter of circulating tumor cells 03 is about 20-30 ⁇ m.
  • the white blood cells 02 and circulating tumor cells 03 slowly aggregate, while the red blood cells 01 are still evenly distributed.
  • the bands of leukocytes 02 and circulating tumor cells 03 aggregates are thinner, and when the liquid influx enters the sorting section 130, the bands of leukocytes 02 and circulating tumor cells 03 aggregates are thinner and closer to the initial stage. Select the inner wall of the runner 10 .
  • the introduction section 110 is set as a reciprocating and folded structure, which can not only play a role of buffering the inflow of blood samples, but also make the liquid flow state more stable, and the blood samples flow through the introduction section 110 and the connecting section 120, which is convenient for circulating tumor cells 03 and leukocytes 02 will gather into bands in this runner.
  • the introduction section 110 and the connecting section 120 are elongated flow channels.
  • the width of the introduction section 110 and the connection section 120 is 0.3mm-1.2mm.
  • the width of the introduction section 110 and the connection section 120 is 0.5mm-0.9mm.
  • the depth of the introduction section 110 and the connection section 120 is 0.06mm-0.3mm.
  • the depth of the introduction section 110 and the connection section 120 is 0.1 mm-0.2 mm.
  • the introduction section 110 includes a plurality of straight pipe sections 112 and a plurality of curved pipe sections 114 , and two adjacent straight pipe sections 112 are connected by a curved pipe section 114 .
  • the pipe section 112 is communicated with the sample inlet 21 through the injection hole 11 , and the most curved pipe section 114 at the end is arranged in a clockwise direction and is connected to the connecting section 120 .
  • This setting makes the introduction section 110 form multiple turns, so that the speed of the blood sample is gradually stabilized, which has a buffering effect and facilitates the initial aggregation of leukocytes 02 and circulating tumor cells 03, and the processing difficulty is easier than the spiral pipeline.
  • the size can be designed to be smaller.
  • the introduction section 110 may also be set in a reciprocatingly folded "S" shape or a spiral shape or the like.
  • the elbow section 114 at the end of the introduction section 110 is connected to the connecting section 120 in a clockwise direction.
  • the main purpose is to ensure that the circulating tumor cells 03 can always accumulate on the side where the inner wall of the flow channel is located during the subsequent sorting process. .
  • the straight pipe section 112 located between the straight pipe section 112 at the beginning and the curved pipe section 114 at the extreme end includes alternately arranged long straight pipes 116 and short straight pipes 118 . Both ends of the long straight pipe 116) are connected to the two short straight pipes 118 through the curved pipe sections 114 arranged in the clockwise direction. Alternatively, both ends of the same long straight pipe 116 (eg, the long straight pipe 116 located above) are connected to the two short straight pipes 118 through the curved pipe sections 114 arranged in the counterclockwise direction. Clockwise and counterclockwise are judged along the flow direction of the sample.
  • the straight pipe sections 112 are arranged in multiple rows in the direction perpendicular to the flow of the sample, and the flow channel positions are arranged reasonably, which is beneficial to reduce the length and width of the chip.
  • long straight tubes 116 and the short straight tubes 118 are arranged in parallel, along the flow direction of the sample, the two adjacent long straight tubes 116 and the short straight tubes 118 located between the two long straight tubes 116 are along the width direction of the straight tube section 112 . spaced arrangement.
  • the long straight pipe 116 and the short straight pipe 118 are connected by a 180° elbow section 114 .
  • the straight pipe section 112 located at the very beginning (the inlet end of the introduction section 110 ) is connected to a long straight pipe 116 through the curved pipe section 114 arranged in the clockwise direction and the curved pipe section 114 arranged in the counterclockwise direction in turn.
  • the straight pipe section 112 located at the extreme end is connected to the connecting section 120 through the curved pipe section 114 arranged in the counterclockwise direction and the curved pipe section 114 arranged in the clockwise direction in turn.
  • the sample flows into the connecting section 120, it flows in a clockwise direction, which is convenient for the circulating tumor cells 03 to be preliminarily aggregated to one side of the inner wall of the flow channel.
  • the connecting section 120 includes a first straight pipe section 121 , a first arc section 122 , a second straight pipe section 123 , a second arc section 124 and a third straight pipe section 121 connected in sequence.
  • Straight pipe section 125 the first straight pipe section 121 is connected to the end of the introduction section 110 away from the sample inlet 21
  • the third straight pipe section 125 is connected to the sorting section 130 .
  • the end of the introduction section 110 is arranged in a clockwise direction and is connected to the first straight pipe section 121 , so that circulating tumor cells 03 always gather on the inner wall of the flow channel.
  • the connection section 120 plays the role of connecting the introduction section 110 and the sorting section 130.
  • the blood sample flows through the first straight pipe section 121, the first arc section 122, the second straight pipe section 123, the first straight pipe section 123, the first straight pipe section 123, the first straight pipe section 122, In the second arc-shaped section 124 and the third straight tube section 125, the bands where the leukocytes 02 and circulating tumor cells 03 gather gradually become thinner.
  • the band is thinner and runs against the inner wall of the pipe.
  • the first arc segment 122 is a 90° arc segment arranged in a clockwise direction
  • the second arc segment 124 is a 90° arc segment arranged in a clockwise direction.
  • the first straight pipe section 121 and the third straight pipe section 125 are arranged at intervals up and down, and the third straight pipe section 125 is located above the first straight pipe section 121 .
  • a first region 126 is formed between the delay line of the first straight pipe section 121 and the delay line of the third straight pipe section 125, and the introduction section 110 is folded back and forth in the range of the first area.
  • the sorting section 130 includes a connecting section 132 and a main pipe section 134 connected in sequence, the connecting section 132 is connected to the third straight pipe section 125 , and the main pipe section 134 Connected to the fine screen flow channel 20, the width of the connecting section 132 perpendicular to its extension direction is a, the width of the main section 134 perpendicular to its extension direction is b, a ⁇ b, and the sorting section 130 is A wavy channel that is asymmetric in the width direction.
  • the width of the flow channel suddenly becomes larger, which disturbs the movement trajectory of the original circulating tumor cells 03, and avoids destroying the original movement of the circulating tumor cells 03 against the inner wall.
  • the connecting section 132 With a width smaller than the main pipe section 134 to transition to the main pipe section 134, it plays a buffering role and avoids the need to flow through more wavy main pipe sections 134 before making the circulating tumor cells 03 close to the inner wall of the flow channel.
  • the length of the main pipe section 134 can also be shortened by forming a thin strip.
  • the connecting section 132 includes alternately arranged first elbow units 13 and second elbow units 14 , and the curvature radius of the first elbow unit 13 is larger than that of the second elbow unit 14 the radius of curvature.
  • the main pipe section 134 includes alternately arranged third elbow units 15 and fourth elbow units 16.
  • the curvature radius of the third elbow unit 15 is greater than the curvature radius of the fourth elbow unit 16.
  • the second elbow unit 14 is connected to the third elbow unit 15 at the first end. Where a is the width of the first elbow unit 13 perpendicular to its extending direction, and b is the width of the third elbow unit 15 perpendicular to its extending direction.
  • the connecting section 132 is formed by alternately connecting the first elbow unit 13 with a large radius of curvature and the second elbow unit 14 with a small radius of curvature.
  • the elbow units 16 are alternately connected and formed.
  • the width of the connecting section 132 perpendicular to its extension direction mainly refers to the width of the first elbow unit 13 perpendicular to its extension direction
  • the width of the main pipe section 134 perpendicular to its extension direction mainly refers to the third elbow unit 15 perpendicular to its extension direction. The width in the extension direction.
  • circulating tumor cells 03 are close to the bottom of the inner wall of the main pipe section 134, and leukocytes 02 are gradually approaching the bottom of the inner wall of the main pipe section 134.
  • b is 0.4mm-1.2mm larger than a.
  • b is 0.7mm-0.9mm larger than a.
  • the first elbow unit 13 includes a first side wall 13a and a second side wall 13b disposed opposite to each other, and the first side wall 13a and the second side wall 13b are asymmetrical curved surfaces.
  • the second elbow unit 14 includes a third side wall 14a and a fourth side wall 14b disposed opposite to each other, and the third side wall 14a and the fourth side wall 14b are asymmetrical curved surfaces.
  • the third side wall 14a is connected to the second side wall 13b, and the fourth side wall 14b is connected to the first side wall 13a.
  • the third elbow unit 15 includes a fifth side wall 15a and a sixth side wall 15b disposed opposite to each other, and the fifth side wall 15a and the sixth side wall 15b are asymmetrical curved surfaces.
  • the fourth elbow unit 16 includes a seventh side wall 16a and an eighth side wall 16b disposed opposite to each other, and the seventh side wall 16a and the eighth side wall 16b are asymmetrical curved surfaces.
  • the fifth side wall 15a is connected to the eighth side wall 16b
  • the sixth side wall 15b is connected to the seventh side wall 16a.
  • the first elbow unit 13 and the second elbow unit 14 are protruded in different directions
  • the third elbow unit 15 and the fourth elbow unit 16 are protruded in different directions
  • the third elbow unit 15 and the fourth elbow unit 16 are protruded in different directions.
  • a pipe bend unit 13 and the third pipe bend unit 15 are protruded in the same direction.
  • the asymmetrical surface is set in this way to form asymmetrical inertial aggregation, so that circulating tumor cells focus on a stable position in the cross-section of the flow channel (the bottom of the inner wall of the flow channel) to form a band, form a focused flow, and flow downstream.
  • the radius of curvature of the first side wall 13a is smaller than the radius of curvature of the second side wall 13b, and the radius of curvature of the fifth side wall 15a is greater than the radius of curvature of the sixth side wall 15b. Therefore, the distance between the first side wall 13a and the second side wall 13b is smaller than the distance between the fifth side wall 15a and the sixth side wall 15b, and the buffering of the fluid from the connecting section 132 to the main pipe section 134 is realized. Further, the radius of curvature of the third side wall 14a is smaller than the radius of curvature of the fourth side wall 14b. The curvature radius of the seventh side wall 16a is smaller than the curvature radius of the eighth side wall 16b.
  • a removal channel 50 is further provided on the first side surface of the functional board 1 .
  • the removal flow channel 50 communicates with the end of the first turning flow channel 40 away from the fine screening flow channel 20 , and the deepened flow channel 30 extends into the removal flow channel 50 correspondingly and is located away from the accumulation of circulating tumor cells 03
  • a diverter hole 51 penetrating the wall surface of the removal flow channel 50 is opened.
  • the blood continues to flow in the removal channel 50 and flows through the shunt hole 51, and the circulating tumor cells 03 continue to move against the bottom of the inner wall of the removal channel 50. Since the white blood cells 02 and red blood cells 01 are distributed evenly in the channel, some white blood cells 02 and red blood cells 01 Then, it flows out from the shunt hole 51 to facilitate subsequent recovery of the circulating tumor cells 03 .
  • the flow rate of the liquid in the flow channel will be relatively low, and the movement trajectory of the circulating tumor cells 03 will change slightly, and it is easy to move in the direction away from the inner wall of the flow channel,
  • the circulating tumor cells 03 are easy to approach the shunt hole 51, and by connecting a first turning flow channel 40 with a large radius of curvature before removing the flow channel 50, the circulating tumor cells 03 flow through the first turning flow channel 40 with a large curvature, and then close to the first turning flow channel 40. It flows against the bottom of the inner wall of the flow channel to prevent circulating tumor cells 03 from flowing into the shunt hole 51, and to improve the recovery rate of circulating tumor cells 03.
  • the second side surface of the functional board 1 is provided with a buffer flow channel 90 in a reciprocating and folded structure, and the buffer flow channel 90 is connected to the shunt hole 51 .
  • the buffer flow channel 90 is designed as a reciprocating and folded structure to stabilize the state of the liquid flowing out of the removal flow channel 50 , so as to prevent the remaining liquid flow in the removal flow channel 50 from being disturbed by jitter, which affects the subsequent recovery of circulating tumor cells 03 .
  • the length of the buffer flow channel 90 can control the amount of discharged liquid.
  • the length of the buffer channel 90 and the position of the shunt hole 51 are used to adjust the amount of leukocytes 02 and erythrocytes 01 discharged and the movement track of the circulating tumor cells 03 .
  • the proportion of leukocytes 02 and erythrocytes 01 removed by the shunt hole 51 is 30%-70%, preferably 45%-60%.
  • the proportion of the liquid flowing out of the shunt hole 51 is 30%-70%, and the optimal range is 45%-60%.
  • the liquid outflow is less than 70%, and the final flow rate is relatively reduced by no more than 70%, so as to avoid affecting the original movement trajectory of circulating tumor cells 03, making it difficult for some circulating tumor cells 03 to be recovered, resulting in a decrease in the final recovery rate. .
  • a blocking member 52 is provided in the removal channel 50 corresponding to the inlet of the shunt hole 51 , and the blocking member 52 is located in the shunt hole 51 away from the deepening
  • the width of the blocking member 52 along the extending direction of the removal flow channel 50 is larger than the diameter of the flow distribution hole 51 .
  • the blocking member 52 protects the circulating tumor cells 03 from flowing into the shunt hole 51. Since the leukocytes 02 and erythrocytes 01 are relatively uniformly distributed in the flow channel, the leukocytes 02 and 01 flow out from the shunt hole 51 and flow vertically into the second side of the functional plate 1 , flows into the buffer flow channel 90 .
  • the blocking member 52 is disposed protruding from the wall surface of the removal flow channel 50, that is, the height of the position where the blocking member 52 is located is higher than other positions of the removal flow channel 50, and is blocked between the circulating tumor cells 03 and the shunt hole 51, The circulating tumor cells 03 are prevented from flowing out of the shunt hole 51 .
  • the side wall of the blocking member 52 close to the accumulation of circulating tumor cells 03 is an arc-shaped wall, and the bending direction of the arc-shaped wall is consistent with the bending direction of the side wall of the removal channel 50 close to the accumulation of circulating tumor cells 03 .
  • the side of the blocking member 52 close to the CTC 03 aggregation is an arc-shaped wall, which is matched with the CTC 03 aggregation band, so that the effect of the blocking member 52 on the CTC 03 mer is consistent and avoids disturbing the circulating tumor cell 03 mer .
  • the blocking member 52 is integrally formed with the functional board 1 , and the blocking member 52 is formed to remove the protruding portion reserved during the opening process of the flow channel 50 .
  • the middle part of one side of the blocking member 52 away from the arc-shaped wall is disposed around the outer circumference of the corresponding side of the diverting hole 51 .
  • the white blood cells 02 and red blood cells 01 flowing to the shunt hole 51 are blocked by the side wall of the blocking member 52 away from the arc-shaped wall, and finally flow into the shunt hole 51 .
  • a plurality of shunt holes 51 are arranged in sequence along the extending direction of the removal channel 50 , and a plurality of reciprocating holes are arranged on the second side of the functional board 1 .
  • the buffer flow channel 90 of the folded-back structure is in one-to-one correspondence with the distribution hole 51 .
  • Multiple shunt holes 51 are arranged along the liquid flow direction to remove leukocytes 02 and erythrocytes 01 multiple times, which is beneficial to the subsequent recovery of circulating tumor cells 03. Since there is no need to aggregate leukocytes 02 and erythrocytes 01, the width and height of the flow channel can also be designed. Wider, which is beneficial to increase the liquid flow rate and improve the sorting efficiency.
  • a blocking member 52 is provided at the inlet of each corresponding distribution hole 51 in the removal flow channel 50 , and the blocking member 52 is located on the side of the distribution hole 51 away from the deepened flow channel 30 .
  • the width of 52 along the extension direction of the removal channel 50 is greater than the diameter of the shunt hole 51, the blocking member 52 protects the circulating tumor cells 03 from flowing into the shunt hole 51, and the white blood cells 02 and red blood cells 01 flow out from the shunt hole 51, It flows vertically into the second side surface of the functional board 1 and flows into the buffer flow channel 90 .
  • the distance L between the plurality of the distribution holes 51 and the corresponding deepened flow channel 30 increases gradually.
  • the white blood cells 02 and the red blood cells 01 will gradually flow to the side away from the deepened flow channel 30.
  • the shunt holes 51 are gradually set away from the deepened flow channel 30, which is convenient for white blood cells. 02 and red blood cells 01 flow out from the subsequent shunt hole 51 .
  • the length of the buffer flow channel 90 corresponding to the distribution hole 51 close to the first turning flow channel 40 is greater than the length of other buffer flow channels 90 .
  • the length of the buffer flow channel 90 corresponding to the first distribution hole 51 is longer than that of the other designs, because the amount of liquid flowing into the first distribution hole 51 will be larger than that of the other distribution holes 51 .
  • the buffer flow channel 90 is designed to be longer, and the buffer flow channel 90 can flow out more liquid, so that the outflowing liquid tends to be stable, so as to avoid affecting the flow rate of the subsequent liquid and disturbing the flow of the circulating tumor cells 03 .
  • the fine-screening flow channel 20 and the removal flow channel 50 are sinusoidal arc-shaped flow channels.
  • the fluid flowing in a parabolic shape has the highest velocity in the middle of the channel.
  • the fluid in the middle of the microchannel is subjected to the largest centrifugal force due to its maximum flow rate, and thus flows to the outer sidewall of the arc-shaped channel.
  • the fluid close to the channel wall has the smallest flow velocity and the least centrifugal force, so it is squeezed by the intermediate high velocity fluid.
  • a pair of counter-rotating and symmetrical vortices are formed, which are located at the upper and lower parts of the channel cross-section, respectively, resulting in the secondary flow of Dean vortices.
  • the Dean vortex exerts a drag force on the particles in the fluid, known as the Dean drag force.
  • the flowing particles will be affected by the inertial lift force and Dean's drag force at the same time, and the relative magnitude of these two forces determines the focused flow of the particles flowing in the arc-shaped channel.
  • the circulating tumor cells 03 are focused into a band on the inner wall of the flow channel.
  • the first side surface of the functional board 1 is further provided with a second turning flow channel 60 .
  • the curvature radius of the second turning flow channel 60 is greater than the curvature radius of the removal flow channel 50, and the second turning flow channel 60 communicates with the end of the removal flow channel 50 away from the first turning flow channel 40,
  • the deepened flow channel 30 correspondingly extends into the second curved flow channel 60 and is located on the side away from the accumulation of circulating tumor cells 03 .
  • the content of white blood cells 02 and red blood cells 01 gradually decreases, and the flow rate also decreases gradually.
  • an end of the second turning flow channel 60 away from the removal flow channel 50 is provided with a recovery flow channel 70 and a waste liquid flow channel 80 that are independent of each other.
  • the recovery flow channel 70 The waste flow channel communicates with the side of the second curved flow channel 60 close to the accumulation of circulating tumor cells 03 , and the waste liquid flow channel communicates with the side of the second curved flow channel 60 close to the deepened flow channel 30 .
  • the circulating tumor cells 03 flow into the recovery flow channel 70 against the inner wall of the second curved flow channel 60 , and the white blood cells 02 and the red blood cells 01 flow into the waste liquid flow channel 80 .
  • the waste liquid flow channel and the recovery flow channel 70 are in a reciprocating and folded structure, which stabilizes the liquid flow state and prevents the liquid from falling from the waste liquid hole 32 and the recovery hole 31.
  • the movement trajectory of the circulating tumor cells 03 at the end of the second turning channel 60 is shown.
  • the volume ratio of the two can be adjusted, thereby adjusting the ratio of the removal of white blood cells 02 and red blood cells 01 .
  • the proportion of the liquid flowing out of the waste liquid flow channel is 45%-65%, preferably 50%-60%.
  • the proportion of the liquid flowing out of the recovery channel 70 is 3%-20%, preferably 5%-10%.
  • the recovery rate of circulating tumor cells 03 can reach over 90%, and the removal rate of white blood cells 02 and red blood cells 01 can reach over 90%.
  • Returning the recovered liquid to the chip for multiple cycles of filtration can further improve the removal rate of white blood cells 02 and red blood cells 01, and improve the recovery purity of circulating tumor cells 03.
  • the upper cover 2 is bonded to the first side of the functional board 1, and the lower cover 3 is bonded to the second side of the functional board 1 , so that each flow channel on the first side and the second side of the functional board 1 forms a sealed channel.
  • the upper cover plate 2 is provided with a sample inlet 21 that communicates with the primary selection flow channel 10 on the first side of the functional board 1 , so that blood samples can be introduced into the microfluidic chip from the sample inlet 21 .
  • the lower cover plate 3 is provided with a recovery hole 31, a waste liquid hole 32 and a discharge hole 33, the shunt hole 51 is communicated with the discharge hole 33, and some leukocytes 02 and erythrocytes 01 flow from the shunt hole 51 to the function in the removal channel 50.
  • the second side of the board 1, and flows out of the microfluidic chip from the discharge hole 33 of the lower cover 3.
  • the recovery channel 70 passes through the first outflow hole 71 through the first side and the second side of the function board.
  • the hole 31 is connected, and the sorted circulating tumor cells 03 flow out of the microfluidic chip through the recovery hole 31 of the lower cover plate 3, and the waste liquid flow channel passes through the second outflow hole 81 through the first side and the second side of the functional board. It communicates with the waste liquid hole 32 , and other remaining liquids flow out of the microfluidic chip through the waste liquid hole 32 of the lower cover plate 3 .
  • the traditional microfluidic chips for CTC 03 sorting and enrichment are basically made of PDMS material.
  • the cost of such chips is expensive and cannot be reused, resulting in thousands of yuan per test.
  • Most of the world's cancer deaths occur in low- and middle-income countries, so it is necessary to develop an inexpensive test.
  • the processing technology of PDMS chips can only design pipes on one side, which limits the development of its functions.
  • the PDMS chip is not conducive to the fabrication of larger and deeper pipes.
  • the multi-level liquid flow state cannot be studied in this chip.
  • the smaller and thinner pipes of PDMS also limit the flow rate of the liquid in the chip.
  • the material of the microfluidic chip in the above embodiment is not limited, and materials such as PMMA, PC, ABS, glass, etc. may be used.
  • the chip can be reused without antigen antibody and magnetic beads, thus greatly reducing the cost. Since this chip only accumulates circulating tumor cells 03, but not white blood cells 02 and red blood cells 01, the height and width of the flow channel in the chip can be larger, and the blood sample can be diluted multiple times, which can make the liquid flow rate larger and reduce detection. time.
  • the design strategy of this chip is: circulating tumor cells 03 gather into thin strips in the chip flow channel, and the flow channel 30 is deepened by design, so that white blood cells 02 and red blood cells 01 are distributed evenly in the chip as much as possible, which is convenient for the recovery of circulating tumor cells 03.
  • the chip design incorporates fundamental principles such as asymmetric inertial focusing and Dean vortices.
  • a multi-level design is adopted, which is conducive to the discharge of white blood cells 02.
  • the chip also adopts a multi-dimensional pipeline design, which is beneficial to reduce the movement trajectory of the leukocytes 02 extruding the circulating tumor cells 03 in the chip.
  • the volume of the recovery channel 70 for recovering circulating tumor cells 03 accounts for a small amount, and the volume of the waste fluid channel accounts for a large proportion. Therefore, leukocytes 02 and erythrocytes 01 can be indirectly removed when recovering tumor cells. Due to the high recovery rate of circulating tumor cells 03 in this chip, it can be filtered multiple times. As a result, the reduction of circulating tumor cells 03 is relatively small on the whole, and the leukocytes 02 are almost completely removed after multiple filtrations.
  • An embodiment of the present application also provides a method for sorting and enriching circulating tumor cells, which can be implemented by using the microfluidic chip for sorting and enriching circulating tumor cells described in any of the above embodiments.
  • the method for sorting and enriching circulating tumor cells in this embodiment includes the following steps:
  • the diluted blood sample is passed through the sample inlet 21 of the microfluidic chip, and after the blood sample passes through the primary selection flow channel 10 with a reciprocating and folded structure, the circulating tumor cells 03 in the blood sample Preliminarily aggregated with leukocytes 02, wherein circulating tumor cells 03 aggregated into thin bands and are close to the bottom of the inner wall of the primary flow channel 10, while the leukocytes 02 have not yet gathered at the bottom of the inner wall of the primary flow channel 10, and the red blood cells 01 are scattered in the primary selection channel within 10.
  • the fine screening flow channel 20 is provided with a deepened flow channel 30 on the side away from the accumulation of circulating tumor cells 03.
  • the circulating tumor cells 03 of the cells gather into thin bands and are close to the bottom of the inner wall of the fine sieve flow channel 20 , while the leukocytes 02 are in a disordered movement state and are far away from the bottom of the inner wall of the fine sieve flow channel 20 .
  • the blood sample After the blood sample is diluted, it is passed through the sample inlet 21 into the primary selection channel 10. Due to the influence of inertial lift, Dean's drag force, etc., the diameter of the red blood cells 01 is small, and the flow in the primary selection channel 10 is chaotic and disorderly. However, the diameters of leukocytes 02 and circulating tumor cells 03 are relatively large.
  • the primary selection channel 10 Under the balance of the forces in the primary selection channel 10, they will initially aggregate into bands, and then flow into the fine screening channel 20, and the circulating tumor cells 03 rigidly aggregate into The thin band is close to the bottom of the inner wall of the fine screening flow channel 20, and the leukocytes 02 have not yet accumulated at the bottom of the inner wall of the tube, but the bands where the leukocytes 02 and the circulating tumor cells 03 are gathered are very close.
  • a deepening flow channel 30 is dug on the side where the tumor cells 03 gather, the deepening flow channel 30 is arranged along the extending direction of the fine screening flow channel 20 and the depth of the deepening flow channel 30 is greater than the depth of the fine screening flow channel 20, Disrupting the liquid flow state near the outer wall of the fine screening flow channel 20 makes the inertial lift force and Dean drag force change, destroying the original balance, so that the leukocyte 02 can produce a disordered movement state, which in turn makes the leukocyte 02 in the fine screening.
  • the distribution in the flow channel 20 is more uniform, avoiding the overlap of the leukocyte 02 aggregate with the circulating tumor cell 03, and the red blood cell 01 is also distributed more evenly, which not only ensures that the aggregate of the circulating tumor cell 03 does not interfere, but also prevents the leukocyte 02 It is accumulated at the bottom of the inner wall of the fine screening flow channel 20, which is convenient for the subsequent separation of the circulating tumor cells 03 and the white blood cells 02, and is conducive to the recovery of the circulating tumor cells 03.
  • the method for sorting and enriching circulating tumor cells further comprises the following steps:
  • circulating tumor cells 03 are easy to approach the shunt hole 51, and by connecting a first turning flow channel 40 with a large radius of curvature before removing the flow channel 50, the circulating tumor cells 03 flow through the first turning flow channel 40 with a large curvature. , and flow close to the bottom of the inner wall of the flow channel to prevent circulating tumor cells 03 from flowing into the shunt hole 51 and improve the recovery rate of circulating tumor cells 03 .
  • the blood sample in the first turning flow channel 40 is introduced into the removal channel 50 having the shunt hole 51, and the red blood cells 01 and leukocyte O2 in the blood sample partially flow out of the removal channel 50 through the shunt hole 51,
  • the circulating tumor cells 03 in the blood sample aggregate into thin bands and are close to the bottom of the inner wall of the removal channel 50 .
  • the blood continues to flow in the removal channel 50 and flows through the shunt hole 51, and the circulating tumor cells 03 continue to move against the bottom of the inner wall of the removal channel 50. Since the white blood cells 02 and red blood cells 01 are distributed evenly in the channel, some white blood cells 02 and red blood cells 01 Then, it flows out from the shunt hole 51 to facilitate subsequent recovery of the circulating tumor cells 03 .
  • the remaining blood sample after the removal of the flow channel 50 is introduced into the second curved flow channel 60 with a radius of curvature greater than that of the removed flow channel 50 , and the circulating tumor cells 03 in the blood sample aggregate into thin strips and further approach the first The bottom of the inner wall of the second turning flow channel 60 .
  • the movement trajectory of the circulating tumor cells 03 can be stabilized, which facilitates the subsequent recovery of the circulating tumor cells 03 .
  • a recovery flow channel 70 is connected to the side of the second turning flow channel 60 close to the accumulation of circulating tumor cells 03, and the blood sample on this side is recovered, and the other side is connected with a waste liquid flow channel, and the blood sample on this side is connected to the recovery flow channel 70. collect.
  • the circulating tumor cells 03 flow into the recovery flow channel 70 against the inner wall of the second curved flow channel 60 , and the white blood cells 02 and the red blood cells 01 flow into the waste liquid flow channel 80 .
  • the method for sorting and enriching circulating tumor cells further comprises the following steps:
  • a blocking member 52 is provided in the removal flow channel 50 corresponding to the inlet of the distribution hole 51 , and the blocking member 52 is located on the side of the distribution hole 51 away from the deepened flow channel 30 , The width of the blocking member 52 along the extending direction of the removal channel 50 is larger than the diameter of the distribution hole 51 .
  • the blocking member 52 blocks between the circulating tumor cells 03 and the shunt hole 51 to prevent the circulating tumor cells 03 from flowing out of the shunt hole 51 .
  • the proportion of blood samples flowing out through the shunt hole 51 is 30%-70%.
  • the outflow of the prepared liquid is less than 70%, and the final flow rate is relatively reduced by no more than 70%, so as to avoid affecting the original movement trajectory of the circulating tumor cells 03, making it difficult for some circulating tumor cells 03 to be recovered. The recovery rate decreased.
  • the proportion of blood samples flowing out through the waste liquid flow channel is 45%-65%.
  • the volume ratio of the two can be adjusted, thereby adjusting the ratio of the removal of white blood cells 02 and red blood cells 01 .
  • the volume of the recovery channel 70 for recovering circulating tumor cells 03 accounts for a small amount, and the volume of the waste liquid channel accounts for a large proportion. Therefore, white blood cells 02 and red blood cells 01 can be indirectly removed when the tumor cells are recovered.
  • an embodiment of the present application further provides an in-vitro analysis and diagnosis instrument, including a main body and the microfluidic chip for sorting and enriching circulating tumor cells according to any of the above embodiments.
  • the enriched microfluidic chip can be used with the main body.
  • the microfluidic chip for sorting and enriching circulating tumor cells belongs to consumables and is used for sorting and enriching circulating tumor cells 03 in blood.
  • the recovered circulating tumor cells 03 can be filtered for multiple cycles to improve the purity of the finally recovered circulating tumor cells 03.
  • the main body is provided with a chip mounting position for installing the microfluidic chip for sorting and enriching circulating tumor cells, and a mixing chamber for mixing the sample, diluent and lysate evenly and a recovery cavity for recycling circulating tumor cells, the mixing cavity can be communicated with the sample inlet of the microfluidic chip for sorting and enriching circulating tumor cells, and the recycling cavity can be sorting and enriching with the circulating tumor cells.
  • the microfluidic chip is connected to the recovery pores flowing out of circulating tumor cells.
  • the in-vitro analysis and diagnosis instrument further includes a power system and a control system, the control system is used to control the power system to inject the sample, the diluent and the lysate into the mixing chamber in a specific proportion, and to inject the mixing chamber into the mixing chamber.
  • the mixed liquid of the body is passed into the microfluidic chip for sorting and enriching the circulating tumor cells at a certain flow rate.
  • the power system can be a pneumatic pump or a syringe pump, etc.
  • the mixing chamber has the function of magnetic stirring or ventilation mixing.
  • control system is also used to control the power system to pass the cleaning solution into the mixing chamber and the microfluidic chip for sorting and enriching the circulating tumor cells in advance, and clean and remove the connecting pipeline, the mixing chamber and the chip. bubble. Then the control system issues a command to control the power system to inject the sample, diluent and lysate into the mixing chamber in a certain proportion, and the liquid in the mixing chamber is passed into the CTC sorting enrichment chamber by the power system at a certain flow rate. set of microfluidic chips. After the mixed liquid of the sample passes through the microfluidic chip for sorting and enriching the circulating tumor cells, the circulating tumor cells are separated into the recovery chamber, and the rest of the liquid enters the waste liquid chamber.
  • the in-vitro analysis and diagnosis instrument further includes a first control valve
  • the main body is further provided with a cleaning fluid chamber, a sample chamber, a diluting fluid chamber and a lysing fluid chamber.
  • the liquid cavity, the sample cavity, the diluent cavity and the lysate cavity are respectively connected with the power system, and one end of the first control valve is connected to the cleaning liquid cavity, the sample cavity, the diluent cavity and the The outlet of the lysate chamber is connected to the mixing chamber, and the other end is connected to the mixing chamber.
  • the power system drives the sample in the sample chamber, the diluent in the diluent chamber, and the lysate in the lysate chamber to flow toward the mixing chamber in a certain proportion, and the first control valve controls the cleaning fluid chamber and the sample chamber. , the on-off of the diluent chamber, the lysate chamber and the mixing chamber.
  • the in vitro analysis and diagnosis instrument further includes a second control valve, one end of the second control valve is connected to the mixing chamber, and the other end is connected to the recovery chamber.
  • the cavity is also connected to the power system.
  • the liquid in the recovery chamber can be sent to the mixing chamber again through the power system and the second control valve, mixed with the diluent again, and re-introduced into the chip for a second time
  • the third and fourth filtering can be performed. High purity circulating tumor can be obtained.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • the terms “installed”, “connected”, “connected”, “fixed” and other terms should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection , or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between the two elements, unless otherwise specified limit.
  • installed may be a fixed connection or a detachable connection , or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between the two elements, unless otherwise specified limit.
  • a first feature "on” or “under” a second feature may be in direct contact between the first and second features, or the first and second features indirectly through an intermediary touch.
  • the first feature being “above”, “over” and “above” the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is level higher than the second feature.
  • the first feature being “below”, “below” and “below” the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature has a lower level than the second feature.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Sustainable Development (AREA)
  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

一种体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法。微流控芯片包括功能板（1），功能板（1）的第一侧面上设有与样本入口连通初选流道（10）及与初选流道（10）连通精筛流道（20），精筛流道（20）中远离循环肿瘤细胞（03）聚集的一侧挖设有加深流道（30），加深流道（30）沿所述精筛流道（20）延伸方向设置且加深流道（30）的深度大于精筛流道（20）的深度。初选流道（10）中作用力的平衡下，循环肿瘤细胞（03）和白细胞（02）会初步聚集成带，而后流入精筛流道（20）内，在精筛流道（20）中远离循环肿瘤细胞聚集的一侧挖设有加深流道（30），打乱精筛流道（20）外壁附近的液体流动状态，使得白细胞（02）能产生无序的运动状态，防止白细胞（02）聚集于精筛流道（20）内壁底部，便于后续把循环肿瘤细胞（03）与白细胞（02）分离开，利于循环肿瘤细胞（03）的回收。

Description

体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法 技术领域
本发明涉及细胞分选技术领域，特别是涉及一种体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法。
背景技术
癌症在全球正常死因中排名第二，每6人中就有1人死于癌症。肿瘤转移是引起癌症90％的死亡原因。肿瘤转移过程就是肿瘤细胞从原发灶或转移灶上脱落，在淋巴系统或外周血中循环，从而入侵远端的组织，并形成新的肿瘤灶，最终导致患者死亡。这些脱落下来的肿瘤细胞就称为循环肿瘤细胞(circulating tumor cell,CTC)。因而通过检查血液中循环肿瘤细胞的数量和种类，即可监测肿瘤病变的动态变化，评估治疗效果。对分离出来的循环肿瘤细胞进行免疫分型、基因组测序等分析，可以找到药物靶点，从而实现个性化精准治疗。循环肿瘤细胞对于肿瘤早期筛查也极为重要，肿瘤在1-2毫米时，影像学等手段很难检测出来。但是在很多中癌症的早期阶段，血液中就含有一定数量的循环肿瘤细胞。对于一些有高风险患者，定期的进行循环肿瘤细胞检测，有助于癌症的早发现，早治疗，避免病情恶化。
因此，从血液中分选和富集循环肿瘤细胞显得尤为重要，但是循环肿瘤细胞含量极低，通常情况下每毫升的血液中含1-10个循环肿瘤细胞，每毫升白细胞的含量为数百万个，红细胞含量为数十亿个，要分选和富集循环肿瘤细胞的难度如同大海捞针。针对分选和富集循环肿瘤细胞的技术大致分为：免疫磁珠法、密度梯度离心法、膜过滤法、微流控芯片技术。其中，传统的微流控芯片的流道循环肿瘤细胞与白细胞容易重叠，不利于循环肿瘤细胞的回收。
发明内容
基于此，有必要针对上述的问题，提供一种体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法，能有效避免循环肿瘤细胞与白细胞重叠，利于循环肿瘤细胞的回收。
一种循环肿瘤细胞分选富集的微流控芯片，包括功能板，所述功能板的第一侧面上设有：
初选流道，与样本入口连通，用于使样本中的循环肿瘤细胞与白细胞初步聚集；及
精筛流道，与所述初选流道连通，所述精筛流道中远离循环肿瘤细胞聚集的一侧挖设有加深流道，所述加深流道沿所述精筛流道延伸方向设置且所述加深流道的深度大于所述精筛流道的深度。
上述循环肿瘤细胞分选富集的微流控芯片，将血液样本稀释后由样本入口通入初选流道，由于惯性升力、迪恩曳力等的受力影响，红细胞的直径较小，在初选流道中混乱无序的流动，而白细胞和循环肿瘤细胞的直径较大，在初选流道中作用力的平衡下，会初步聚集成带，而后流入精筛流道内，循环肿瘤细胞刚聚体成细带，并靠近精筛流道内壁底部，而白细胞还未聚集于管道内壁底部，但白细胞和循环肿瘤细胞聚集的带靠得很近，通过在精筛流道中远离循环肿瘤细胞聚集的一侧挖设有加深流道，加深流道沿所述精筛流道延伸方向设置且所述加深流道的深度大于所述精筛流道的深度，打乱了精筛流道外壁附近的液体流动状态，使得惯性升力与迪恩曳力改变，破坏原有的平衡，从而使得白细胞能产生无序的运动状态，进而使得白细胞在精筛流道中分布更加均匀，避免白细胞聚体的带和循环肿瘤细胞重叠，同理红细胞也分布得更加均匀，既保证了不干扰循环肿瘤细胞的聚体，也防止了白细胞聚集于精筛流道内壁底部，便于后续把循环肿瘤细胞与白细胞分离开，利于循环肿瘤细胞的回收。
在其中一实施例中，所述加深流道的深度比所述精筛流道的深度大50μm-200μm；或者所述加深流道的深度比所述精筛流道的深度大70μm-120μm。
在其中一实施例中，所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比小于等于0.5；或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比小于等于0.07；或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比为0.045～0.065；或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比为0.05～0.06。
在其中一实施例中，所述功能板的第一侧面上还设有第一转弯流道，所述第一转弯流道的曲率半径大于所述精筛流道的曲率半径，所述第一转弯流道与所述精筛流道流通，所述加深流道对应地延伸至所述第 一转弯流道中且位于远离循环肿瘤细胞聚集的一侧。
在其中一实施例中，所述功能板的第一侧面上还设有去除流道，所述去除流道与所述第一转弯流道远离精筛流道的一端连通，所述加深流道对应地延伸至所述去除流道中且位于远离循环肿瘤细胞聚集的一侧，所述去除流道中开设有贯穿所述去除流道壁面的分流孔。
在其中一实施例中，所述功能板的第一侧面上还设有第二转弯流道，所述第二转弯流道的曲率半径大于所述去除流道的曲率半径，所述第二转弯流道与所述去除流道远离所述第一转弯流道的一端连通，所述加深流道对应地延伸至所述第二转弯流道中且位于远离循环肿瘤细胞聚集的一侧。
在其中一实施例中，所述第二转弯流道远离所述去除流道的一端设有相互独立的回收流道与废液流道，所述回收流道与所述第二转弯流道中靠近循环肿瘤细胞聚集的一侧连通，所述废液流道与所述第二转弯流道中靠近加深流道的一侧连通。
在其中一实施例中，所述废液流道与所述回收流道流出的液体的占比为45％-65％：3％-20％；或者所述废液流道与所述回收流道流出的液体的占比为50％-60％：5％-10％。
在其中一实施例中，所述废液流道与所述回收流道呈往复回折结构。
在其中一实施例中，所述功能板的第二侧面上设有呈往复回折结构的缓冲流道，所述缓冲流道与所述分流孔连通。
在其中一实施例中，所述缓冲流道流出的液体的占比为30％-70％；或者所述缓冲流道流出的液体的占比为45％-60％。
在其中一实施例中，所述去除流道中对应所述分流孔入口处设有阻挡件，且所述阻挡件位于所述分流孔远离所述加深流道的一侧，所述阻挡件沿所述去除流道的延伸方向的宽度大于所述分流孔的直径。
在其中一实施例中，沿所述去除流道的延伸方向依次设有多个分流孔，所述功能板的第二侧面上设有多个呈往复回折结构的缓冲流道，所述缓冲流道与所述分流孔一一对应连通；所述去除流道中对应所述分流孔入口处设有阻挡件，且所述阻挡件位于所述分流孔远离所述加深流道的一侧，所述阻挡件沿所述去除流道的延伸方向的宽度大于所述分流孔的直径。
在其中一实施例中，沿样本流动方向，多个所述分流孔与对应的所述加深流道的距离逐渐增大。
在其中一实施例中，沿样本流动方向，靠近所述第一转弯流道的分流孔对应的缓冲流道的长度大于其他缓冲流道的长度。
在其中一实施例中，所述初选流道包括依次连通的导入段、连接段及分选段，所述导入段远离所述连接段的一端与所述样本入口连通，所述导入段呈往复回折结构。
在其中一实施例中，所述连接段包括依次连通的第一直管段、第一弧形段、第二直管段、第二弧形段及第三直管段，第一直管段与所述导入段远离样本入口的一端连接，所述第三直管段与所述分选段连接。
在其中一实施例中，所述导入段的末端沿顺时针方向设置且与所述第一直管段连接。
在其中一实施例中，所述分选段包括依次连通的衔接段与主管段，所述衔接段与所述第三直管段连接，所述主管段与所述精筛流道连接，所述衔接段垂直其延伸方向上的宽度为a，所述主管段垂直其延伸方向上的宽度为b，a<b，所述分选段为宽度方向上不对称的波浪形通道。
在其中一实施例中，所述精筛流道及所述去除流道为正弦型弧形流道。
在其中一实施例中，所述的循环肿瘤细胞分选富集的微流控芯片还包括上盖板与下盖板，所述上盖板上开设有样本入口，且所述上盖板与所述功能板的第一侧面叠置连接，所述下盖板上开设有回收孔、废液孔及排出孔，且所述下盖板与所述功能板的第二侧面叠置连接，所述回收流道与所述回收孔连通，所述废液流道与所述废液孔连通，所述分流孔与所述排出孔连通。
一种循环肿瘤细胞分选富集的方法，包括如下步骤：
将经稀释后的血液样本由微流控芯片的样本入口通入，血液样本经过具有往复回折结构的初选流道后，血液样本中的循环肿瘤细胞与白细胞初步聚集，其中循环肿瘤细胞聚集成细带并靠近初选流道内壁底部，而白细胞还未聚集于初选流道内壁底部，红细胞分散于所述初选流道内；
将所述初选流道中的血液样本导入与精筛流道，所述精筛流道中远离循环肿瘤细胞聚集的一侧挖设有加深流道，血液样本中的循环肿瘤细胞聚集成细带并靠近精筛流道内壁底部，而白细胞处于无序运动状态且远离所述精筛流道内壁底部。
上述循环肿瘤细胞分选富集的方法，将血液样本稀释后由样本入口通入初选流道，由于惯性升力、迪恩曳力等的受力影响，红细胞的直径较小，在初选流道中混乱无序的流动，而白细胞和循环肿瘤细胞的直径较大，在初选流道中作用力的平衡下，会初步聚集成带，而后流入精筛流道内，循环肿瘤细胞刚聚体成细带，并靠近精筛流道内壁底部，而白细胞还未聚集于管道内壁底部，但白细胞和循环肿瘤细胞聚集的带靠得很近，通过在精筛流道中远离循环肿瘤细胞聚集的一侧挖设有加深流道，加深流道沿所述精筛流道延伸方向设置且所述加深流道的深度大于所述精筛流道的深度，打乱了精筛流道外壁附近的液体流动状态，使得惯性升力与迪恩曳力改变，破坏原有的平衡，从而使得白细胞能产生无序的运动状态，进而使得白细胞在精筛流道中分布更加均匀，避免白细胞聚体的带和循环肿瘤细胞重叠，同理红细胞也分布得更加均匀，既保证了不干扰循环肿瘤细胞的聚体，也防止了白细胞聚集于精筛流道内壁底部，便于后续把循环肿瘤细胞与白细胞分离开，利于循环肿瘤细胞的回收。
在其中一实施例中，循环肿瘤细胞分选富集的方法还包括如下步骤：
将所述精筛流道中的血液样本导入曲率半径大于所述精筛流道的曲率半径的第一转弯流道内，血液样本中的循环肿瘤细胞聚集成细带进一步靠近第一转弯流道内壁底部；
将所述第一转弯流道中的血液样本导入具有分流孔的去除流道，血液样本中的红细胞与白细胞部分由所述分流孔流出所述去除流道，而血液样本中的循环肿瘤细胞聚集成细带并靠近去除流道内壁底部；
经所述去除流道后剩余的血液样本导入曲率半径大于所述去除流道的曲率半径的第二转弯流道，血液样本中的循环肿瘤细胞聚集成细带进一步靠近第二转弯流道内壁底部；
第二转弯流道靠近循环肿瘤细胞聚集的一侧连通有回收流道并对该侧的血液样本进行回收，另一侧连通有废液流道并对该侧的血液样本进行收集。
所述的循环肿瘤细胞分选富集的方法还包括如下步骤：
将所述回收流道回收的血液样本再次通入微流控芯片的样本入口，重复前述各步骤。
在其中一实施例中，所述的循环肿瘤细胞分选富集的方法，其中所述去除流道中对应所述分流孔入口处设有阻挡件，且所述阻挡件位于所述分流孔远离所述加深流道的一侧，所述阻挡件沿所述去除流道的延伸方向的宽度大于所述分流孔的直径,经所述分流孔流出的血液样本占比为30％-70％；经所述废液流道流出的血液样本占比为45％-65％。
一种体外分析诊断仪，包括主体及上述任一项所述的循环肿瘤细胞分选富集的微流控芯片，所述循环肿瘤细胞分选富集的微流控芯片能与所述主体配套使用。
在其中一实施例中，所述主体上设有用于安装所述循环肿瘤细胞分选富集的微流控芯片的芯片安装位、用于将样本、稀释液及裂解液混匀的混合腔体以及回收循环肿瘤细胞的回收腔体，所述混合腔体能与所述循环肿瘤细胞分选富集的微流控芯片的样本入口连通，所述回收腔体能与所述循环肿瘤细胞分选富集的微流控芯片中流出循环肿瘤细胞的回收孔连通。
在其中一实施例中，所述的体外分析诊断仪还包括动力系统及控制系统，所述控制系统用于控制动力系统将样本、稀释液及裂解液按特定比例注入混合腔体以及将混合腔体的混合液体以一定的流速通入所述循环肿瘤细胞分选富集的微流控芯片中。
在其中一实施例中，所述的体外分析诊断仪还包括第一控制阀，所述主体上还设有清洗液腔体、样本腔体、稀释液腔体及裂解液腔体，所述清洗液腔体、样本腔体、稀释液腔体及裂解液腔体分别与所述动力系统连接，所述第一控制阀的一端与所述清洗液腔体、样本腔体、稀释液腔体及裂解液腔体的出口连接，另一端与所述混合腔体连接。
在其中一实施例中，所述的体外分析诊断仪还包括第二控制阀，所述第二控制阀的一端与所述混合腔体连接，另一端与所述回收腔体连接，所述回收腔体还与所述动力系统连接。
附图说明
图1为一实施例中循环肿瘤细胞分选富集的微流控芯片的上盖板的示意图；
图2为一实施例中循环肿瘤细胞分选富集的微流控芯片的功能板的第一侧面的示意图；
图3为一实施例中循环肿瘤细胞分选富集的微流控芯片的功能板的第二侧面的示意图；
图4为一实施例中循环肿瘤细胞分选富集的微流控芯片的下盖板的示意图；
图5为图2中沿剖面线A-A的剖视示意图；
图6为血液样本中循环肿瘤细胞、白细胞、红细胞在初选流道的导入段与连接段中的运动状态示意图；
图7为血液样本中循环肿瘤细胞、白细胞、红细胞在初选流道的分选段中的运动状态示意图；
图8为血液样本中循环肿瘤细胞、白细胞、红细胞在第一转弯流道中的运动状态示意图；
图9为血液样本中循环肿瘤细胞、白细胞、红细胞在去除流道中的运动状态示意图；
图10为血液样本中循环肿瘤细胞、白细胞、红细胞在第二转弯流道中的运动状态示意图；
图11为一实施例中体外分析诊断仪的示意图。
附图标记说明：
01、红细胞；02、白细胞；03、循环肿瘤细胞；1、功能板；10、初选流道；11、进样孔；110、导入段；112、直管段；114、弯管段，116、长直管；118、短直管，120、连接段；121、第一直管段；122、第一弧形段；123、第二直管段；124、第二弧形段；125、第三直管段；130、分选段；132、衔接段；13、第一弯管单元；13a、第一侧壁；13b、第二侧壁；14、第二弯管单元，14a、第三侧壁；14b、第四侧壁；134、主管段；15、第三弯管单元；第五侧壁15a与第六侧壁15b，16、第四弯管单元，16a、第七侧壁；16b、第八侧壁；20、精筛流道；30、加深流道；40、第一转弯流道；50、去除流道；51、分流孔；52、阻挡件；60、第二转弯流道；70、回收流道；71、第一流出孔；80、废液流道；81、第二流出孔；90、缓冲流道；2、上盖板；21、样本入口；3、下盖板；31、回收孔；32、废液孔；33、排出孔。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施例的限制。
请参阅图1-4，一实施例提供一种循环肿瘤细胞分选富集的微流控芯片，包括功能板1。进一步地，循环肿瘤细胞分选富集的微流控芯片还包括上盖板2与下盖板3。所述上盖板2与所述功能板1的第一侧面叠置连接，所述下盖板3与所述功能板1的第二侧面叠置连接，所述上盖板2上开设有样本入口21。
参照图2、6、7，功能板1的第一侧面上设有初选流道10与精筛流道20。初选流道10与样本入口21连通，用于使样本中的循环肿瘤细胞03与白细胞02初步聚集。精筛流道20与所述初选流道10连通，所述精筛流道20中远离循环肿瘤细胞03聚集的一侧挖设有加深流道30，所述加深流道30沿所述精筛流道20延伸方向设置且所述加深流道30的深度大于所述精筛流道20的深度。
将血液样本稀释后由上盖板2的样本入口21通入微流控芯片内，经功能板1上的进样孔11流入初选流道10。其中，进样孔11贯穿功能板1的第一侧面与第二侧面，且与样本入口21对应设置。由于惯性升力、迪恩曳力等的受力影响，参照图6，样本中的红细胞01的直径较小，在初选流道10中混乱无序的流动，而白细胞02和循环肿瘤细胞03的直径较大，在初选流道10中作用力的平衡下，会初步聚集成带再流入精筛流道20内，参照图7，此时循环肿瘤细胞03刚聚体成细带，并靠近精筛流道20内壁底部，而白细胞02还未聚集于流道内壁底部，但白细胞02和循环肿瘤细胞03聚集的带靠得很近，通过在精筛流道20中远离循环肿瘤细胞03聚集的一侧挖设有加深流道30，加深流道30沿所述精筛流道20延伸方向设置且所述加深流道30的深度大于所述精筛流道20的深度，打乱了精筛流道20外壁附近的液体流动状态，使得惯性升力与迪恩曳力改变，破坏原有的平衡，从而使得白细胞02能产生无序的运动状态，进而使得白细胞02在精筛流道20中分布更加均匀，避免白细胞02聚体的带和循环肿瘤细胞03重叠，同理红细胞01也分布得更加均匀，既保证了不干扰循环肿瘤细胞03的聚体，也防止了白细胞02聚集于精筛流道20内壁底部，便于后续把循环肿瘤细胞03与白细胞02分离开，利于循环肿瘤细胞03的回收。
参照图2、7，在其中一实施例中，所述精筛流道为不对称波浪形通道，其具有交替布设的大转弯和小转弯。在其他实施例中，所述精筛流道也可设置为直通道。
具体地，在其中一实施例中，所述精筛流道为具有矩形截面的不对称波浪形通道，该矩形截面的宽高比在通道的长度范围内变化导致形成单条聚集粒子流。
可选地，所述矩形截面的宽高比在8～30之间变化。在另一个实施例中，所述矩形截面的宽高比在11～23之间变化。
可选地，在其中一实施例中，所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比小于等于0.5。或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比小于等于0.07。或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比为0.045～0.065。或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比为0.05～0.06。其中，水力直径Dh定义为2wh/(w+h),参照图5、7；w和h为流道的宽度和高度。
进一步地，参照图2、8，在其中一实施例中，所述功能板1的第一侧面上还设有第一转弯流道40。所述第一转弯流道40的曲率半径大于所述精筛流道20的曲率半径，所述第一转弯流道40与所述精筛流道20流通，参照图5，所述加深流道30对应地延伸至所述第一转弯流道40中且位于远离循环肿瘤细胞03聚集的一侧。在加深流道30的作用下，使得白细胞02在第一转弯流道40中分布更加均匀，避免与循环肿瘤细胞03重叠。在精筛流道20连接了一个第一转弯流道40，由于第一转弯流道40的半径大，惯性升力的相对优势更加大。经过第一转弯流道40处时,流道中间流体受到的离心力最大,从而流向通道外侧边缘。靠近通道壁的流体流速最小,所受离心力也最小,从而受到中间流体的挤压。从而在第一转弯流道40的作用下，循环肿瘤细胞03能大限度地贴近管道内壁底部，为后续分选循环肿瘤细胞03和排出白细胞02做准备。
参照图2、5，可选地，所述加深流道30的深度比所述精筛流道20的深度大50μm-200μm。可选地，精筛流道的深度为100μm-200μm。优选地，所述加深流道30的深度比所述精筛流道20的深度大70μm-120μm。优选地，精筛流道的深度为110μm-150μm。图5为加深流道30在第一转弯流道40中的截面示意图，其中精筛流道20延伸至与第一转弯流道40连接，第一转弯流道40中对应也开设有加深流道30，加深流道30与第一转弯流道40的深度差H为50μm-200μm，优先70μm-120μm。既能保证循环肿瘤细胞03在精筛流道20或第一转弯流道40中能尽可能地靠近流道内壁底部流动，同时扰动加深流道30一侧的液体流动状态，避免白细胞02聚体的带和循环肿瘤细胞03重叠而影响后续循环肿瘤细胞03的回收率及纯度。
参照图2、6，在其中一实施例中，所述初选流道10包括依次连通的导入段110、连接段120及分选段130，所述导入段110远离所述连接段120的一端与所述样本入口21连通，所述导入段110呈往复回折结构。参照图6，样本进入导入段110的入口端时，红细胞01、白细胞02和循环肿瘤细胞03均匀的分布在流道中。红细胞01直径约为6-8μm和白细胞02直径约为8-12μm，循环肿瘤细胞03直径约为20-30μm。当液体流过导入段110的几个拐弯处时，白细胞02和循环肿瘤细胞03慢慢聚集，而红细胞01还是均匀的分布。随着流过越长的连接段120，白细胞02和循环肿瘤细胞03聚集的带越细，当液体流入快进入分选段130时，白细胞02和循环肿瘤细胞03聚集的带更加细，并且靠初选流道10的内壁。将导入段110设置为往复回折结构，不仅能起到缓冲流入血液样本的作用，可以使得液体流动状态更加稳定，而且血液样本流过导入段110及连接段120，便于循环肿瘤细胞03和白细胞02会在此流道中聚集成带。
相比分选段130，导入段110与连接段120为细长的流道。可选地，导入段110、连接段120的宽度为0.3mm-1.2mm。优选地，导入段110、连接段120的宽度为0.5mm-0.9mm。可选地，导入段110、连接段120的深度为0.06mm-0.3mm。优选地，导入段110、连接段120的深度为0.1mm-0.2mm。如此设置，使进入初选流道10的血液样本流速趋于平稳，同时使循环肿瘤细胞03和白细胞02能初步聚集成带，便于后续对循环肿瘤细胞03进行分选。
具体地，参照图6，一实施例中所述导入段110包括多个直管段112及多个弯管段114，两相邻直管段112之间通过一个弯管段114连接，最始端的直管段112通过进样孔11与样本入口21连通，最末端的弯管段114沿顺时针方向设置且与所述连接段120连接。如此设置使导入段110形成多个拐弯，使血液样本速度逐渐趋于平稳，起到缓冲效果，便于白细胞02和循环肿瘤细胞03初步聚集，加工难度相较螺旋形管道也更容易，芯片的整体尺寸可设计得更小。在其他实施例中，为实现缓冲的目的，导入段110也可设置成往复回折的“S”形或者螺旋形等。其中导入段110最末端的弯管段114沿顺时针方向设置与所述连接段120连接，主要目的是为了使后续分选过程中循环肿瘤细胞03能始终聚集于流道的内壁所在的一侧。
在一实施例中，位于最始端的直管段112与最末端的弯管段114之间的直管段112包括交替设置的长直管116与短直管118，同一长直管116(如位于下方的长直管116)的两端均通过沿顺时针方向设置的弯 管段114与两短直管118连接。或者同一长直管116(如位于上方的长直管116)的两端均通过沿逆时针方向设置的弯管段114与两短直管118连接。其中顺时针、逆时针是沿样本流动方向判断的。通过交替设置长直管116与短直管118，使直管段112在垂直样本流动的方向上呈多排设置，合理布置流道位置，利于缩小芯片的长度与宽度。
进一步地，所述长直管116与短直管118平行设置，沿样本流动方向，相邻两长直管116与位于该两长直管116之间的短直管118沿直管段112宽度方向间隔布置。长直管116与短直管118之间通过180°的弯管段114连接，长直管116与短直管118平行设置，在沿直管段112宽度方向直管段112成三排布置。
具体地，参照图6，位于最始端(导入段110的入口端)直管段112依次通过顺时针方向设置的弯管段114与逆时针方向设置的弯管段114与一长直管116连接。位于最末端直管段112依次通过逆时针方向设置的弯管段114与顺时针方向设置的弯管段114与所述连接段120连接。样本流入连接段120时呈顺时针方向流动，便于循环肿瘤细胞03初步聚集成带于流道的内壁一侧。
参照图2、6，在其中一实施例中，所述连接段120包括依次连通的第一直管段121、第一弧形段122、第二直管段123、第二弧形段124及第三直管段125，第一直管段121与所述导入段110远离样本入口21的一端连接，所述第三直管段125与所述分选段130连接。具体地，所述导入段110的末端沿顺时针方向设置且与所述第一直管段121连接，使循环肿瘤细胞03始终聚集在流道的内壁。连接段120起到连接导入段110与分选段130的作用，血液样本由导入段110末端沿顺时针方向依次流经第一直管段121、第一弧形段122、第二直管段123、第二弧形段124及第三直管段125，白细胞02和循环肿瘤细胞03聚集的带逐渐变细，当液体位于第三直管段125快进入分选段130时，白细胞02和循环肿瘤细胞03聚集的带更加细，并且靠管道的内壁。在其中一实施例中，所述第一弧形段122为沿顺时针方向设置的90°弧形段，所述第二弧形段124为沿顺时针方向设置的90°弧形段。第一直管段121与第三直管段125上下间隔设置，且第三直管段125位于第一直管段121上方。
进一步地，所述第一直管段121的延迟线与第三直管段125的延迟线之间围设形成第一区域126,所述导入段110在所述第一区域的范围内往复回折。通过合理布置流道各段的位置，充分利用芯片的体积，便于使芯片整体尺寸小型化，也便于白细胞02和循环肿瘤细胞03初步聚集成带。
参照图2、7，在其中一实施例中，所述分选段130包括依次连通的衔接段132与主管段134，所述衔接段132与所述第三直管段125连接，所述主管段134与所述精筛流道20连接，所述衔接段132垂直其延伸方向上的宽度为a，所述主管段134垂直其延伸方向上的宽度为b，a<b，所述分选段130为宽度方向上不对称的波浪形通道。为避免血液样本从细长的连接段120进入波浪形的分选段130时，流道的宽度突然变大较多，扰乱原来循环肿瘤细胞03的运动轨迹，避免破坏循环肿瘤细胞03原来靠内壁运动的轨迹，通过设置宽度小于主管段134的衔接段132过渡到主管段134，起到缓冲作用，避免需要流过更多的波浪形的主管段134后才能使得循环肿瘤细胞03贴近流道内壁聚集成细带，也能缩短主管段134的长度。
参照图7，具体地，所述衔接段132包括交替设置的第一弯管单元13与第二弯管单元14，所述第一弯管单元13的曲率半径大于所述第二弯管单元14的曲率半径。所述主管段134包括交替设置的第三弯管单元15与第四弯管单元16，所述第三弯管单元15的曲率半径大于所述第四弯管单元16的曲率半径,最末端的第二弯管单元14与最始端的所述第三弯管单元15连接。其中a为所述第一弯管单元13垂直其延伸方向上的宽度，b为所述第三弯管单元15垂直其延伸方向上的宽度。衔接段132通过曲率半径大的第一弯管单元13与曲率半径小的第二弯管单元14交替连接形成，主管段134通过曲率半径大的第三弯管单元15与曲率半径小的第四弯管单元16交替连接形成。所述衔接段132垂直其延伸方向上的宽度主要指第一弯管单元13垂直其延伸方向上的宽度，所述主管段134垂直其延伸方向上的宽度主要指第三弯管单元15垂直其延伸方向上的宽度。根据细胞直径、流道高度和宽度、液体流速等因素，综合惯性升力、迪恩曳力等的受力分析，循环肿瘤细胞03靠近主管段134内壁底部，白细胞02也逐渐靠近主管段134内壁底部。可选地，b比a大0.4mm-1.2mm。优选地，b比a大0.7mm-0.9mm。
参照图7，进一步地，第一弯管单元13包括相对设置的第一侧壁13a与第二侧壁13b，所述第一侧壁13a与第二侧壁13b为非对称的曲面。第二弯管单元14包括相对设置的第三侧壁14a与第四侧壁14b，所述第三侧壁14a与第四侧壁14b为非对称的曲面。所述第三侧壁14a与所述第二侧壁13b连接，所述第四侧壁14b与所述第一侧壁13a连接。所述第三弯管单元15包括相对设置的第五侧壁15a与第六侧壁15b， 所述第五侧壁15a与第六侧壁15b为非对称的曲面。第四弯管单元16包括相对设置的第七侧壁16a与第八侧壁16b，所述第七侧壁16a与第八侧壁16b为非对称的曲面。所述第五侧壁15a与所述第八侧壁16b连接，所述第六侧壁15b与第七侧壁16a连接。所述第一弯管单元13与第二弯管单元14朝不同的方向凸出设置，所述第三弯管单元15与第四弯管单元16朝不同的方向凸出设置，且所述第一弯管单元13与所述第三弯管单元15朝相同的方向凸出设置。如此设置非对称曲面，形成非对称惯性聚集，使循环肿瘤细胞在流道横截面中稳定位置(流道的内壁底部)聚焦形成一个带，形成聚焦流动，流向下游。
进一步地，所述第一侧壁13a的曲率半径小于所述第二侧壁13b的曲率半径，所述第五侧壁15a的曲率半径大于第六侧壁15b的曲率半径。从而使第一侧壁13a与第二侧壁13b之间的距离小于第五侧壁15a与第六侧壁15b之间的距离，实现流体由衔接段132到主管段134的缓冲。进一步地，所述第三侧壁14a的曲率半径小于所述第四侧壁14b的曲率半径。所述第七侧壁16a的曲率半径小于所述第八侧壁16b的曲率半径。
进一步地，参照图2、9，在其中一实施例中，所述功能板1的第一侧面上还设有去除流道50。所述去除流道50与所述第一转弯流道40远离精筛流道20的一端连通，所述加深流道30对应地延伸至所述去除流道50中且位于远离循环肿瘤细胞03聚集的一侧，所述去除流道50中开设有贯穿所述去除流道50壁面的分流孔51。血液继续在去除流道50中流动，流经分流孔51，循环肿瘤细胞03继续贴着去除流道50内壁底部运动，由于白细胞02和红细胞01在管道中分布较为均匀，部分白细胞02和红细胞01则从分流孔51流出，便于后续循环肿瘤细胞03的回收。
参照图2、8、9，由于随着液体从分流孔51流出，流道中液体的流速相对会变低，循环肿瘤细胞03运动轨迹会发生微小的变化，容易向着远离流道内壁的方向运动，循环肿瘤细胞03易靠近分流孔51，而通过在去除流道50之前衔接一个曲率半径大的第一转弯流道40，使循环肿瘤细胞03流过大弯的第一转弯流道40后，紧贴着流道内壁底部流动，避免循环肿瘤细胞03流入分流孔51，提高循环肿瘤细胞03的回收率。
参照图2、3，进一步地，在其中一实施例中，所述功能板1的第二侧面上设有呈往复回折结构的缓冲流道90，所述缓冲流道90与所述分流孔51连通。将缓冲流道90设计为往复回折结构，使流出去除流道50的液体状态稳定，避免去除流道50中剩余液体流动受到抖动干扰，影响后续循环肿瘤细胞03的回收。
其中，缓冲流道90的长短可以控制排出液体的多少。缓冲流道90的长度及结合分流孔51的位置来调节排出白细胞02和红细胞01的量及调控循环肿瘤细胞03的运动轨迹。一实施例中，分流孔51去除的白细胞02和红细胞01占比为30％-70％，优选45％-60％。同理分流孔51所流出的液体占比为30％-70％，最优范围为45％-60％。如此设置，使液体流出量小于70％，最后流速相对减小不超过70％以上，避免影响到循环肿瘤细胞03原来的运动轨迹，造成部分循环肿瘤细胞03难以被回收，导致最后的回收率下降。
参照图2、9，在其中一实施例中，所述去除流道50中对应所述分流孔51入口处设有阻挡件52，且所述阻挡件52位于所述分流孔51远离所述加深流道30的一侧，所述阻挡件52沿所述去除流道50的延伸方向的宽度大于所述分流孔51的直径。通过阻挡件52保护循环肿瘤细胞03不被流入分流孔51，由于白细胞02和红细胞01在流道中分布较为均匀，白细胞02和红细胞01则从分流孔51流出，垂直流入功能板1的第二侧面，流入缓冲流道90。
进一步地，所述阻挡件52凸出所述去除流道50的壁面设置，即阻挡件52所在位置的高度高于去除流道50其他位置，阻挡在循环肿瘤细胞03与分流孔51之间，避免循环肿瘤细胞03从分流孔51流出。所述阻挡件52靠近循环肿瘤细胞03聚集的一侧为弧形壁，且所述弧形壁的弯曲方向与所述去除流道50靠近循环肿瘤细胞03聚集的一侧侧壁的弯曲方向一致。阻挡件52靠近循环肿瘤细胞03聚集的一侧为弧形壁，与循环肿瘤细胞03聚集带匹配，使阻挡件52对循环肿瘤细胞03聚体的影响保持一致，避免扰乱循环肿瘤细胞03聚体。
可选地，所述阻挡件52与所述功能板1为一体成型结构，阻挡件52为去除流道50开设过程中预留的凸出部分形成。所述阻挡件52远离弧形壁的一侧中部绕所述分流孔51对应侧的外周设置。流向分流孔51的白细胞02与红细胞01被阻挡件52远离弧形壁的侧壁挡住，最终流入分流孔51。
参照图2、3、9，在其中一实施例中，沿所述去除流道50的延伸方向依次设有多个分流孔51，所述功能板1的第二侧面上设有多个呈往复回折结构的缓冲流道90，所述缓冲流道90与所述分流孔51一一对 应连通。沿液体流动方向设置多个分流孔51对白细胞02和红细胞01进行多次去除，有利于后续循环肿瘤细胞03的回收，由于无需聚集白细胞02与红细胞01，流道的宽度和高度也可以设计得较宽，利于提高液体流速，提高分选效率。同时在所述去除流道50中各对应的分流孔51入口处设有阻挡件52，且所述阻挡件52位于所述分流孔51远离所述加深流道30的一侧，所述阻挡件52沿所述去除流道50的延伸方向的宽度大于所述分流孔51的直径，通过阻挡件52保护循环肿瘤细胞03不被流入分流孔51，白细胞02和红细胞01则从分流孔51流出，垂直流入功能板1的第二侧面，流入缓冲流道90。
参照图9，在其中一实施例中，沿样本流动方向，多个所述分流孔51与对应的所述加深流道30的距离L逐渐增大。随着液体的排出，白细胞02和红细胞01则会逐渐向远离加深流道30的一侧流动，对应沿样本流动方向，将所述分流孔51逐渐向远离加深流道30的方向设置，便于白细胞02和红细胞01从后续分流孔51流出。
在其中一实施例中，沿样本流动方向，靠近所述第一转弯流道40的分流孔51对应的缓冲流道90的长度大于其他缓冲流道90的长度。参照图3可以看出，第一个分流孔51对应的缓冲流道90的长度较其他的设计得更长，由于第一个分流孔51流入的液体量相对其他的分流孔51会更多，将缓冲流道90设计得更长，缓冲流道90能流出更多的液体，使流出的液体趋于平稳，避免对后续液体的流速造成影响，扰乱循环肿瘤细胞03的流动。
在其中一实施例中，所述精筛流道20及所述去除流道50为正弦型弧形流道。流体在弧形通道中流动时，呈抛物线流动的流体在通道中间速度最大。在经过通道转弯处时，微通道中间的流体因其流速最大而受到的离心力最大，从而流向弧形通道的外侧壁。靠近通道壁的流体流速最小，所受离心力也最小，从而受到中间高流速流体的挤压。为了保持流体中各处质量守恒，在垂直于流体流动的方向上，形成一对反向旋转且对称的涡流，分别位于通道横截面的上部和下部，由此产生迪恩涡流的二次流。迪恩涡流会对流体中的颗粒产生曳力作用，被称为迪恩曳力。在弧形通道中，流动的粒子会同时受到惯性升力和迪恩曳力的作用，这两种力的相对大小决定粒子在弯弧形通道中流动的聚焦流动情况。本实施例中，精筛流道20及去除流道50中由于惯性升力和迪恩曳力的作用，循环肿瘤细胞03在流道的内壁聚焦成一带。
进一步地，参照图2、10，在其中一实施例中，所述功能板1的第一侧面上还设有第二转弯流道60。所述第二转弯流道60的曲率半径大于所述去除流道50的曲率半径，所述第二转弯流道60与所述去除流道50远离所述第一转弯流道40的一端连通，所述加深流道30对应地延伸至所述第二转弯流道60中且位于远离循环肿瘤细胞03聚集的一侧。血液流过分流孔51后，白细胞02和红细胞01含量逐渐减少，同时流速也逐渐减小。通过衔接曲率半径大于所述去除流道50的曲率半径的第二转弯流道60，能起到稳定循环肿瘤细胞03的运动轨迹的作用，便于后续对循环肿瘤细胞03的回收。
参照图10，在其中一实施例中，所述第二转弯流道60远离所述去除流道50的一端设有相互独立的回收流道70与废液流道80，所述回收流道70与所述第二转弯流道60中靠近循环肿瘤细胞03聚集的一侧连通，所述废液流道与所述第二转弯流道60中靠近加深流道30的一侧连通。循环肿瘤细胞03紧贴第二转弯流道60内壁流入回收流道70，白细胞02和红细胞01流入废液流道80。在其中一实施例中，所述废液流道与所述回收流道70呈往复回折结构，起到稳定液体流动状态，防止液体后续从废液孔32、回收孔31跌落的过程中，影响了第二转弯流道60末端循环肿瘤细胞03的运动轨迹。
通过调节废液流道、回收流道70的长度和深度，可以调节两者的体积占比，从而调节去除白细胞02和红细胞01占比。在其中一实施例中，废液流道流出的液体占比为45％-65％，优先50％-60％。回收流道70流出的液体占比为3％-20％，优先为5％-10％。从回收孔31收集到的液体，循环肿瘤细胞03回收率可达到90％以上，白细胞02和红细胞01去除率在90％以上。将回收的液体再返回芯片循环过滤多次，能进一步提高白细胞02和红细胞01去除率，提高循环肿瘤细胞03的回收纯度。
参照图1-4，在其中一实施例中，所述上盖板2与所述功能板1的第一侧面粘接，所述下盖板3与所述功能板1的第二侧面粘接，使功能板1的第一侧面与第二侧面的各流道形成密封的通道。所述上盖板2上开设与功能板1的第一侧面的初选流道10连通的样本入口21，便于血液样本从样本入口21导入微流控芯片内。下盖板3上开设有回收孔31、废液孔32及排出孔33，所述分流孔51与所述排出孔33连通，部分白细胞02和红细胞01在去除流道50由分流孔51流向功能板1的第二侧面，并从下盖板3的排出孔33流出微流控芯片，所述回收流道70通过贯穿功能板第一侧面与第二侧面的第一流出孔71与所述回收孔31 连通，分选出的循环肿瘤细胞03由下盖板3的回收孔31流出微流控芯片，所述废液流道通过贯穿功能板第一侧面与第二侧面的第二流出孔81与所述废液孔32连通，剩余的其他液体由下盖板3的废液孔32流出微流控芯片。
传统进行循环肿瘤细胞03分选和富集的微流控芯片基本都是PDMS材质，这种芯片的成本昂贵，不能重复使用，导致每个测试的费用在数千元。但是全球大多数的癌症死亡都发生在中低收入国家，所以开发一款成本低廉的检测产品是很有必要。PDMS芯片的加工工艺只能在单面设计管道，从而限制了其功能的开发。并且PDMS芯片不利于制作较大和较深的管道，多层次的液体流动状态在此芯片中不能研究，PDMS较小较细的管道也限制了芯片中液体的流速。
上述实施例中的微流控芯片材质不受限定，可以用PMMA、PC、ABS、玻璃等材质。本芯片可以重复使用，不需要抗原抗体，也不需要磁珠，从而大大的降低了成本。由于本芯片只聚集循环肿瘤细胞03，不聚集白细胞02和红细胞01，所以芯片中流道的高度和宽度可以较大，血液样本可以做大倍数的稀释，从而可以使得液体流速较大，也减少检测时间。
本芯片的设计策略为：循环肿瘤细胞03在芯片流道中聚集成细带，通过设计加深流道30，使白细胞02、红细胞01尽量在芯片中均匀分配，便于循环肿瘤细胞03的回收。芯片设计结合了不对称的惯性聚焦和迪恩涡旋等基本原理。同时采取了多层次的设计，有利于白细胞02的排出。芯片也采用多维的管道设计，有利于减少白细胞02挤压循环肿瘤细胞03在芯片中的运动轨迹。
另外，回收循环肿瘤细胞03的回收流道70的体积占比很少，废液流道的体积占比很大，因此就可以在回收肿瘤细胞的时候，间接去除了白细胞02和红细胞01。由于此芯片的循环肿瘤细胞03回收率高，所以可以多次过滤。从而整体上循环肿瘤细胞03的减少量较少，白细胞02在多次过滤后，几乎全部去除。
本申请一实施例还提供一种循环肿瘤细胞分选富集的方法，可采用上述任一实施例所述的循环肿瘤细胞分选富集的微流控芯片实现。本实施例的循环肿瘤细胞分选富集的方法包括如下步骤：
参照图1-4，S100：将经稀释后的血液样本由微流控芯片的样本入口21通入，血液样本经过具有往复回折结构的初选流道10后，血液样本中的循环肿瘤细胞03与白细胞02初步聚集，其中循环肿瘤细胞03聚集成细带并靠近初选流道10内壁底部，而白细胞02还未聚集于初选流道10内壁底部，红细胞01分散于所述初选流道10内。
S200：将所述初选流道10中的血液样本导入与精筛流道20，所述精筛流道20中远离循环肿瘤细胞03聚集的一侧挖设有加深流道30，血液样本中的循环肿瘤细胞03聚集成细带并靠近精筛流道20内壁底部，而白细胞02处于无序运动状态且远离所述精筛流道20内壁底部。
将血液样本稀释后由样本入口21通入初选流道10，由于惯性升力、迪恩曳力等的受力影响，红细胞01的直径较小，在初选流道10中混乱无序的流动，而白细胞02和循环肿瘤细胞03的直径较大，在初选流道10中作用力的平衡下，会初步聚集成带，而后流入精筛流道20内，循环肿瘤细胞03刚聚体成细带，并靠近精筛流道20内壁底部，而白细胞02还未聚集于管道内壁底部，但白细胞02和循环肿瘤细胞03聚集的带靠得很近，通过在精筛流道20中远离循环肿瘤细胞03聚集的一侧挖设有加深流道30，加深流道30沿所述精筛流道20延伸方向设置且所述加深流道30的深度大于所述精筛流道20的深度，打乱了精筛流道20外壁附近的液体流动状态，使得惯性升力与迪恩曳力改变，破坏原有的平衡，从而使得白细胞02能产生无序的运动状态，进而使得白细胞02在精筛流道20中分布更加均匀，避免白细胞02聚体的带和循环肿瘤细胞03重叠，同理红细胞01也分布得更加均匀，既保证了不干扰循环肿瘤细胞03的聚体，也防止了白细胞02聚集于精筛流道20内壁底部，便于后续把循环肿瘤细胞03与白细胞02分离开，利于循环肿瘤细胞03的回收。
在其中一实施例中，循环肿瘤细胞分选富集的方法还包括如下步骤：
S300、将所述精筛流道20中的血液样本导入曲率半径大于所述精筛流道20的曲率半径的第一转弯流道40内，血液样本中的循环肿瘤细胞03聚集成细带进一步靠近第一转弯流道40内壁底部。参照图2、8、9，由于后续S400步骤液体会从分流孔51流出，流道中液体的流速相对会变低，循环肿瘤细胞03运动轨迹会发生微小的变化，容易向着远离流道内壁的方向运动，循环肿瘤细胞03易靠近分流孔51，而通过在去除流道50之前衔接一个曲率半径大的第一转弯流道40，使循环肿瘤细胞03流过大弯的第一转弯流道40后，紧贴着流道内壁底部流动，避免循环肿瘤细胞03流入分流孔51，提高循环肿瘤细胞03的回收率。
S400、将所述第一转弯流道40中的血液样本导入具有分流孔51的去除流道50，血液样本中的红细胞01与白细胞02部分由所述分流孔51流出所述去除流道50，而血液样本中的循环肿瘤细胞03聚集成细带并靠近去除流道50内壁底部。血液继续在去除流道50中流动，流经分流孔51，循环肿瘤细胞03继续贴着去除流道50内壁底部运动，由于白细胞02和红细胞01在管道中分布较为均匀，部分白细胞02和红细胞01则从分流孔51流出，便于后续循环肿瘤细胞03的回收。
S500、经所述去除流道50后剩余的血液样本导入曲率半径大于所述去除流道50的曲率半径的第二转弯流道60，血液样本中的循环肿瘤细胞03聚集成细带进一步靠近第二转弯流道60内壁底部。通过衔接曲率半径大于所述去除流道50的曲率半径的第二转弯流道60，能起到稳定循环肿瘤细胞03的运动轨迹的作用，便于后续对循环肿瘤细胞03的回收。
S600、第二转弯流道60靠近循环肿瘤细胞03聚集的一侧连通有回收流道70并对该侧的血液样本进行回收，另一侧连通有废液流道并对该侧的血液样本进行收集。循环肿瘤细胞03紧贴第二转弯流道60内壁流入回收流道70，白细胞02和红细胞01流入废液流道80。
在其中一实施例中，所述的循环肿瘤细胞分选富集的方法还包括如下步骤：
S700、将所述回收流道70回收的血液样本再次通入微流控芯片的样本入口21，重复前述各步骤S100-S600。回收的液体重复前述各步骤S100-S600循环过滤，能进一步提高白细胞02和红细胞01去除率，提高循环肿瘤细胞03的回收纯度。
在其中一实施例中，在去除流道50中对应所述分流孔51入口处设有阻挡件52，且所述阻挡件52位于所述分流孔51远离所述加深流道30的一侧，所述阻挡件52沿所述去除流道50的延伸方向的宽度大于所述分流孔51的直径。阻挡件52阻挡在循环肿瘤细胞03与分流孔51之间，避免循环肿瘤细胞03从分流孔51流出。
进一步地，经所述分流孔51流出的血液样本占比为30％-70％。通过控制液体流出量，使制液体流出量小于70％，最后流速相对减小不超过70％以上，避免影响到循环肿瘤细胞03原来的运动轨迹，造成部分循环肿瘤细胞03难以被回收，导致最后的回收率下降。
进一步地，经所述废液流道流出的血液样本占比为45％-65％。通过调节废液流道、回收流道70的长度和深度，可以调节两者的体积占比，从而调节去除白细胞02和红细胞01占比。回收循环肿瘤细胞03的回收流道70的体积占比很少，废液流道的体积占比很大，因此就可以在回收肿瘤细胞的时候，间接去除了白细胞02和红细胞01。
参照图11，本申请一实施例还提供一种体外分析诊断仪，包括主体及上述任一项实施例所述的循环肿瘤细胞分选富集的微流控芯片，所述循环肿瘤细胞分选富集的微流控芯片能与所述主体配套使用。所述的循环肿瘤细胞分选富集的微流控芯片属于耗材，用于分选及富集血液中的循环肿瘤细胞03。对于同一血液样本，可对回收的循环肿瘤细胞03进行多次循环过滤，以提高最后回收的循环肿瘤细胞03的纯度。
在其中一实施例中，所述主体上设有用于安装所述循环肿瘤细胞分选富集的微流控芯片的芯片安装位、用于将样本、稀释液及裂解液混匀的混合腔体以及回收循环肿瘤细胞的回收腔体，所述混合腔体能与所述循环肿瘤细胞分选富集的微流控芯片的样本入口连通，所述回收腔体能与所述循环肿瘤细胞分选富集的微流控芯片中流出循环肿瘤细胞的回收孔连通。
在其中一实施例中，所述的体外分析诊断仪还包括动力系统及控制系统，所述控制系统用于控制动力系统将样本、稀释液及裂解液按特定比例注入混合腔体以及将混合腔体的混合液体以一定的流速通入所述循环肿瘤细胞分选富集的微流控芯片中。动力系统可以是气压泵或注射泵等。混合腔体具备磁力搅拌或通气混匀功能。进一步地，控制系统还用于控制动力系统预先把清洗液通入混合腔体和所述循环肿瘤细胞分选富集的微流控芯片，对连接管路、混合腔体及芯片进行清洗和排除气泡。然后控制系统再发出命令，控制动力系统把样本和稀释液、裂解液按一定的比例注入混合腔体，混匀腔体的液体被动力系统以一定的流速通入所述循环肿瘤细胞分选富集的微流控芯片。样本的混合液通过所述循环肿瘤细胞分选富集的微流控芯片后，循环肿瘤细胞就被分离进入回收腔体，其余的液体进入废液腔体。
在其中一实施例中，所述的体外分析诊断仪还包括第一控制阀，所述主体上还设有清洗液腔体、样本腔体、稀释液腔体及裂解液腔体，所述清洗液腔体、样本腔体、稀释液腔体及裂解液腔体分别与所述动力系统连接，所述第一控制阀的一端与所述清洗液腔体、样本腔体、稀释液腔体及裂解液腔体的出口连接， 另一端与所述混合腔体连接。动力系统驱动样本腔体中的样本和稀释液腔体中的稀释液、裂解液腔体中的裂解液按一定的比例朝混合腔体流动，第一控制阀控制清洗液腔体、样本腔体、稀释液腔体及裂解液腔体与混合腔体的通断。
在其中一实施例中，所述的体外分析诊断仪还包括第二控制阀，所述第二控制阀的一端与所述混合腔体连接，另一端与所述回收腔体连接，所述回收腔体还与所述动力系统连接。对于复杂的样本或需要纯度较高的样本，回收腔体的液体可以通过动力系统和第二控制阀被再次送入混合腔体中，再次和稀释液混匀，重新通入芯片进行第二次过滤，同理可以进行第三、四次过滤。可以得到纯度高的循环肿瘤。
在本发明的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”、“内”、“外”、“顺时针”、“逆时针”、“轴向”、“径向”、“周向”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系，除非另有明确的限定。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本发明中，除非另有明确的规定和限定，第一特征在第二特征“上”或“下”可以是第一和第二特征直接接触，或第一和第二特征通过中间媒介间接接触。而且，第一特征在第二特征“之上”、“上方”和“上面”可是第一特征在第二特征正上方或斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”可以是第一特征在第二特征正下方或斜下方，或仅仅表示第一特征水平高度小于第二特征。
需要说明的是，当元件被称为“固定于”或“设置于”另一个元件，它可以直接在另一个元件上或者也可以存在居中的元件。当一个元件被认为是“连接”另一个元件，它可以是直接连接到另一个元件或者可能同时存在居中元件。本文所使用的术语“垂直的”、“水平的”、“上”、“下”、“左”、“右”以及类似的表述只是为了说明的目的，并不表示是唯一的实施方式。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (30)

  1. 一种循环肿瘤细胞分选富集的微流控芯片，其特征在于，包括功能板，所述功能板的第一侧面上设有：
    初选流道，与样本入口连通，用于使样本中的循环肿瘤细胞与白细胞初步聚集；及
    精筛流道，与所述初选流道连通，所述精筛流道中远离循环肿瘤细胞聚集的一侧挖设有加深流道，所述加深流道沿所述精筛流道延伸方向设置且所述加深流道的深度大于所述精筛流道的深度。
  2. 根据权利要求1所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述加深流道的深度比所述精筛流道的深度大50μm-200μm；或者所述加深流道的深度比所述精筛流道的深度大70μm-120μm。
  3. 根据权利要求1所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比小于等于0.5；或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比小于等于0.07；或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比为0.045～0.065；或者所述精筛流道设计为循环肿瘤细胞的尺寸与水力直径之比为0.05～0.06。
  4. 根据权利要求1所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述功能板的第一侧面上还设有第一转弯流道，所述第一转弯流道的曲率半径大于所述精筛流道的曲率半径，所述第一转弯流道与所述精筛流道流通，所述加深流道对应地延伸至所述第一转弯流道中且位于远离循环肿瘤细胞聚集的一侧。
  5. 根据权利要求4所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述功能板的第一侧面上还设有去除流道，所述去除流道与所述第一转弯流道远离精筛流道的一端连通，所述加深流道对应地延伸至所述去除流道中且位于远离循环肿瘤细胞聚集的一侧，所述去除流道中开设有贯穿所述去除流道壁面的分流孔。
  6. 根据权利要求5所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述功能板的第一侧面上还设有第二转弯流道，所述第二转弯流道的曲率半径大于所述去除流道的曲率半径，所述第二转弯流道与所述去除流道远离所述第一转弯流道的一端连通，所述加深流道对应地延伸至所述第二转弯流道中且位于远离循环肿瘤细胞聚集的一侧。
  7. 根据权利要求6所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述第二转弯流道远离所述去除流道的一端设有相互独立的回收流道与废液流道，所述回收流道与所述第二转弯流道中靠近循环肿瘤细胞聚集的一侧连通，所述废液流道与所述第二转弯流道中靠近加深流道的一侧连通。
  8. 根据权利要求7所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述废液流道与所述回收流道流出的液体的占比为45％-65％：3％-20％；或者所述废液流道与所述回收流道流出的液体的占比为50％-60％：5％-10％。
  9. 根据权利要求7所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述废液流道与所述回收流道呈往复回折结构。
  10. 根据权利要求5-9任一项所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述功能板的第二侧面上设有呈往复回折结构的缓冲流道，所述缓冲流道与所述分流孔连通。
  11. 根据权利要求10所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述缓冲流道流出的液体的占比为30％-70％；或者所述缓冲流道流出的液体的占比为45％-60％。
  12. 根据权利要求5-9任一项所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述去除流道中对应所述分流孔入口处设有阻挡件，且所述阻挡件位于所述分流孔远离所述加深流道的一侧，所述阻挡件沿所述去除流道的延伸方向的宽度大于所述分流孔的直径。
  13. 根据权利要求5-9任一项所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，沿所述去除流道的延伸方向依次设有多个分流孔，所述功能板的第二侧面上设有多个呈往复回折结构的缓冲流道，所述缓冲流道与所述分流孔一一对应连通；所述去除流道中对应所述分流孔入口处设有阻挡件，且所述阻挡件位于所述分流孔远离所述加深流道的一侧，所述阻挡件沿所述去除流道的延伸方向的宽度大于所述分流孔的直径。
  14. 根据权利要求13所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，沿样本流动方向，多个所述分流孔与对应的所述加深流道的距离逐渐增大。
  15. 根据权利要求13所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，沿样本流动方向，靠近所述第一转弯流道的分流孔对应的缓冲流道的长度大于其他缓冲流道的长度。
  16. 根据权利要求1-9任一项所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述初选流道包括依次连通的导入段、连接段及分选段，所述导入段远离所述连接段的一端与所述样本入口连通，所述导入段呈往复回折结构。
  17. 根据权利要求16所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述连接段包括依次连通的第一直管段、第一弧形段、第二直管段、第二弧形段及第三直管段，第一直管段与所述导入段远离样本入口的一端连接，所述第三直管段与所述分选段连接。
  18. 根据权利要求17所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述导入段的末端沿顺时针方向设置且与所述第一直管段连接。
  19. 根据权利要求17所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述分选段包括依次连通的衔接段与主管段，所述衔接段与所述第三直管段连接，所述主管段与所述精筛流道连接，所述衔接段垂直其延伸方向上的宽度为a，所述主管段垂直其延伸方向上的宽度为b，a<b，所述分选段为宽度方向上不对称的波浪形通道。
  20. 根据权利要求5-9任一项所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，所述精筛流道及所述去除流道为正弦型弧形流道。
  21. 根据权利要求7-9任一项所述的循环肿瘤细胞分选富集的微流控芯片，其特征在于，还包括上盖板与下盖板，所述上盖板上开设有样本入口，且所述上盖板与所述功能板的第一侧面叠置连接，所述下盖板上开设有回收孔、废液孔及排出孔，且所述下盖板与所述功能板的第二侧面叠置连接，所述回收流道与所述回收孔连通，所述废液流道与所述废液孔连通，所述分流孔与所述排出孔连通。
  22. 一种循环肿瘤细胞分选富集的方法，其特征在于，包括如下步骤：
    将经稀释后的血液样本由微流控芯片的样本入口通入，血液样本经过具有往复回折结构的初选流道后，血液样本中的循环肿瘤细胞与白细胞初步聚集，其中循环肿瘤细胞聚集成细带并靠近初选流道内壁底部，而白细胞还未聚集于初选流道内壁底部，红细胞分散于所述初选流道内；
    将所述初选流道中的血液样本导入与精筛流道，所述精筛流道中远离循环肿瘤细胞聚集的一侧挖设有加深流道，血液样本中的循环肿瘤细胞聚集成细带并靠近精筛流道内壁底部，而白细胞处于无序运动状态且远离所述精筛流道内壁底部。
  23. 根据权利要求22所述的循环肿瘤细胞分选富集的方法，其特征在于，还包括如下步骤：
    将所述精筛流道中的血液样本导入曲率半径大于所述精筛流道的曲率半径的第一转弯流道内，血液样本中的循环肿瘤细胞聚集成细带进一步靠近第一转弯流道内壁底部；
    将所述第一转弯流道中的血液样本导入具有分流孔的去除流道，血液样本中的红细胞与白细胞部分由所述分流孔流出所述去除流道，而血液样本中的循环肿瘤细胞聚集成细带并靠近去除流道内壁底部；
    经所述去除流道后剩余的血液样本导入曲率半径大于所述去除流道的曲率半径的第二转弯流道，血液样本中的循环肿瘤细胞聚集成细带进一步靠近第二转弯流道内壁底部；
    第二转弯流道靠近循环肿瘤细胞聚集的一侧连通有回收流道并对该侧的血液样本进行回收，另一侧连通有废液流道并对该侧的血液样本进行收集。
  24. 根据权利要求23所述的循环肿瘤细胞分选富集的方法，其特征在于，还包括如下步骤：
    将所述回收流道回收的血液样本再次通入微流控芯片的样本入口，重复前述各步骤。
  25. 根据权利要求23或24所述的循环肿瘤细胞分选富集的方法，其特征在于，其中所述去除流道中对应所述分流孔入口处设有阻挡件，且所述阻挡件位于所述分流孔远离所述加深流道的一侧，所述阻挡件沿所述去除流道的延伸方向的宽度大于所述分流孔的直径,经所述分流孔流出的血液样本占比为30％-70％；经所述废液流道流出的血液样本占比为45％-65％。
  26. 一种体外分析诊断仪，其特征在于，包括主体及权利要求1-21任一项所述的循环肿瘤细胞分选富集的微流控芯片，所述循环肿瘤细胞分选富集的微流控芯片能与所述主体配套使用。
  27. 根据权利要求26所述的体外分析诊断仪，其特征在于，所述主体上设有用于安装所述循环肿瘤细胞分选富集的微流控芯片的芯片安装位、用于将样本、稀释液及裂解液混匀的混合腔体以及回收循环肿 瘤细胞的回收腔体，所述混合腔体能与所述循环肿瘤细胞分选富集的微流控芯片的样本入口连通，所述回收腔体能与所述循环肿瘤细胞分选富集的微流控芯片中流出循环肿瘤细胞的回收孔连通。
  28. 根据权利要求27所述的体外分析诊断仪，其特征在于，还包括动力系统及控制系统，所述控制系统用于控制动力系统将样本、稀释液及裂解液按特定比例注入混合腔体以及将混合腔体的混合液体以一定的流速通入所述循环肿瘤细胞分选富集的微流控芯片中。
  29. 根据权利要求28所述的体外分析诊断仪，其特征在于，还包括第一控制阀，所述主体上还设有清洗液腔体、样本腔体、稀释液腔体及裂解液腔体，所述清洗液腔体、样本腔体、稀释液腔体及裂解液腔体分别与所述动力系统连接，所述第一控制阀的一端与所述清洗液腔体、样本腔体、稀释液腔体及裂解液腔体的出口连接，另一端与所述混合腔体连接。
  30. 根据权利要求28或29所述的体外分析诊断仪，其特征在于，还包括第二控制阀，所述第二控制阀的一端与所述混合腔体连接，另一端与所述回收腔体连接，所述回收腔体还与所述动力系统连接。
PCT/CN2022/073892 2021-01-29 2022-01-26 体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法 WO2022161371A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US18/263,397 US20240084233A1 (en) 2021-01-29 2022-01-26 In vitro analysis diagnostic instrument, and microfluidic chip and method for sorting and enriching circulating tumor cells
EP22745245.5A EP4286506A1 (en) 2021-01-29 2022-01-26 In vitro analysis diagnostic instrument, circulating tumor cell sorting and enrichment micro-fluidic chip and method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110128093.6A CN114806799A (zh) 2021-01-29 2021-01-29 体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法
CN202110128093.6 2021-01-29

Publications (1)

Publication Number Publication Date
WO2022161371A1 true WO2022161371A1 (zh) 2022-08-04

Family

ID=82525303

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/073892 WO2022161371A1 (zh) 2021-01-29 2022-01-26 体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法

Country Status (4)

Country Link
US (1) US20240084233A1 (zh)
EP (1) EP4286506A1 (zh)
CN (1) CN114806799A (zh)
WO (1) WO2022161371A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024067354A1 (zh) * 2022-09-27 2024-04-04 深圳赛桥生物创新技术有限公司 磁珠去除方法、设备和存储介质

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090014360A1 (en) * 2007-04-16 2009-01-15 The General Hospital Corporation D/B/A Massachusetts General Hospital Systems and methods for particle focusing in microchannels
CN104797340A (zh) * 2012-09-21 2015-07-22 麻省理工学院 微流体装置及其用途
CN105854967A (zh) * 2016-06-15 2016-08-17 广东工业大学 一种微流控芯片装置及其微流道结构
US20170333902A1 (en) * 2016-05-19 2017-11-23 The Board Of Trustees Of The Leland Stanford Junior University Systems and Methods for Automated Single Cell Cytological Classification in Flow
CN107699478A (zh) * 2017-09-19 2018-02-16 朱嗣博 一种循环肿瘤细胞（ctc）检测用微流控芯片装置
CN109082368A (zh) * 2018-10-29 2018-12-25 上海理工大学 循环肿瘤细胞分选、富集及检测用多级微流控芯片装置
CN110106069A (zh) * 2019-05-30 2019-08-09 合肥工业大学 一种基于流体动力学的免标记分离癌细胞的生物芯片
CN111330656A (zh) * 2020-03-03 2020-06-26 东南大学 一种微米颗粒悬液体积浓缩微流控器件
CN111763606A (zh) * 2020-06-18 2020-10-13 上海交通大学 从血液中无标记分离循环肿瘤细胞的惯性聚焦微流控芯片
CN112547145A (zh) * 2020-11-19 2021-03-26 东南大学 一种稀有细胞快速筛选微流控器件

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090014360A1 (en) * 2007-04-16 2009-01-15 The General Hospital Corporation D/B/A Massachusetts General Hospital Systems and methods for particle focusing in microchannels
CN104797340A (zh) * 2012-09-21 2015-07-22 麻省理工学院 微流体装置及其用途
US20170333902A1 (en) * 2016-05-19 2017-11-23 The Board Of Trustees Of The Leland Stanford Junior University Systems and Methods for Automated Single Cell Cytological Classification in Flow
CN105854967A (zh) * 2016-06-15 2016-08-17 广东工业大学 一种微流控芯片装置及其微流道结构
CN107699478A (zh) * 2017-09-19 2018-02-16 朱嗣博 一种循环肿瘤细胞（ctc）检测用微流控芯片装置
CN109082368A (zh) * 2018-10-29 2018-12-25 上海理工大学 循环肿瘤细胞分选、富集及检测用多级微流控芯片装置
CN110106069A (zh) * 2019-05-30 2019-08-09 合肥工业大学 一种基于流体动力学的免标记分离癌细胞的生物芯片
CN111330656A (zh) * 2020-03-03 2020-06-26 东南大学 一种微米颗粒悬液体积浓缩微流控器件
CN111763606A (zh) * 2020-06-18 2020-10-13 上海交通大学 从血液中无标记分离循环肿瘤细胞的惯性聚焦微流控芯片
CN112547145A (zh) * 2020-11-19 2021-03-26 东南大学 一种稀有细胞快速筛选微流控器件

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG LEI, DAVID S. DANDY: "High-Throughput Inertial Focusing of Micrometer- and Sub-Micrometer-Sized Particles Separation", ADVANCED SCIENCE, vol. 4, no. 10, 30 May 2017 (2017-05-30), pages 1700153, XP055955044, DOI: 10.1002/advs.201700153 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024067354A1 (zh) * 2022-09-27 2024-04-04 深圳赛桥生物创新技术有限公司 磁珠去除方法、设备和存储介质

Also Published As

Publication number Publication date
EP4286506A1 (en) 2023-12-06
CN114806799A (zh) 2022-07-29
US20240084233A1 (en) 2024-03-14

Similar Documents

Publication Publication Date Title
CN109580323B (zh) 一种螺旋形微通道及其使用方法与串、并联安装结构
US8276760B2 (en) Serpentine structures for continuous flow particle separations
JP6314220B2 (ja) 細胞の検出及び単離のためのマイクロ流体選別器
US8841135B2 (en) Biochip for high-throughput screening of circulating tumor cells
WO2022161371A1 (zh) 体外分析诊断仪、循环肿瘤细胞分选富集的微流控芯片及方法
CN214881401U (zh) 微流控流道及微流控芯片
CN107164213A (zh) 一种基于惯性原理分离细胞的芯片
WO2021013066A1 (zh) 一种适用于循环肿瘤细胞捕获的微流控芯片
CN112354573B (zh) 阶跃式惯性聚焦微流控芯片
WO2022068648A1 (zh) 一种微流控芯片盒
EP3408217A1 (en) Multi-stage target cell enrichment using a microfluidic device
US20200171488A1 (en) Multi-Dimensional Double Spiral Device and Methods of Use Thereof
WO2022161373A1 (zh) 生物粒子分选流道及微流控芯片
TW201927353A (zh) 用於血液樣本之細胞分離的微流體裝置及方法
CN107488582A (zh) 微流控装置
MX2014003296A (es) Metodos para segregar particulas usando un aparato con un elemento de separacion por discriminacion de tamaño que tiene un borde delantero alargado.
CN214881402U (zh) 微流控分选流道及微流控芯片
KR102432895B1 (ko) 미세유체 버블 트랩
WO2022161372A1 (zh) 微流控芯片
CN110205244A (zh) 高通量微流控芯片及其用于分选癌细胞的方法
CN114260037B (zh) 一种3d堆叠式多级惯性微流控分选芯片及其制备方法
CN220143418U (zh) 微流控芯片
RU2782098C1 (ru) Способ улучшения разделения микрожидкостных частиц и устройство для способа
CN114798015A (zh) 微流控芯片
Bagi et al. Size-Based Microparticle Separation Via Inertial Lift and Dean Flow in a Spiral Microchannel Device

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22745245

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 18263397

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2022745245

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022745245

Country of ref document: EP

Effective date: 20230829